pFind Studio: a computational solution for mass spectrometry-based proteomics

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Citations Last Updated: Oct. 1, 2024

2024




Proteome-wide Ligand and Target Discovery by Using Strain-Enabled Cyclopropane Electrophiles
Journal of the American Chemical Society. 2024. Yue Liu et al. State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
ABSTRACT:
Use: pChem



Catalyst-free late-stage functionalization to assemble $\alpha$-acyloxyenamide electrophiles for selectively profiling conserved lysine residues
Communications chemistry. 2024. Zhao, Yuanyuan et al. State Key Laboratory of Bioactive Molecules and Druggability Assessment, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
ABSTRACT:
Use: pChem; pQuant



Structural insights into the cross-exon to cross-intron spliceosome switch
Nature. 2024. Zhang, Zhenwei et al. Cellular Biochemistry, Max-Planck-Institute for Multidisciplinary Sciences, Göttingen, Germany
ABSTRACT: Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5'splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal Bcomplex6,7.
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Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure
Nature Communications. 2024. Michael, Andrew RM et al. Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N-4N1, Canada
ABSTRACT:
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Dynamic molecular architecture and substrate recruitment of cullin3--RING E3 ligase CRL3KBTBD2
Nature Structural & Molecular Biology. 2024. Hu, Yuxia et al. Molecular Genetics II, Center of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141, Essen, Germany
ABSTRACT: Phosphatidylinositol 3-kinase alpha, a heterodimer of catalytic p110 alpha and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85 alpha subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85 alpha ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85 alpha availability.
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Dynamic shape-shifting of the single-celled eukaryotic predator Lacrymaria via unconventional cytoskeletal components
Current biology : CB. 2024. Qin, Weiwei et al. Institute of Hydrobiology, Chinese Academy of Sciences, No. 7 Donghu South Road, Wuchang District, Wuhan 430072, Hubei, China
ABSTRACT: Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "neck" that can stretch 7-8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space search and prey capture.
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Ribosome External Electric Field Regulates Metabolic Enzyme Activity: The RAMBO Effect
JOURNAL OF PHYSICAL CHEMISTRY B. 2024. Yu, Jianchao et al. Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222, United States
ABSTRACT: Ribosomes bind to many metabolic enzymes and change their activity. A general mechanism for ribosome-mediated amplification of metabolic enzyme activity, RAMBO, was formulated and elucidated for the glycolytic enzyme triosephosphate isomerase, TPI. The RAMBO effect results from a ribosome-dependent electric field-substrate dipole interaction energy that can increase or decrease the ground state of the reactant and product to regulate catalytic rates. NMR spectroscopy was used to determine the interaction surface of TPI binding to ribosomes and to measure the corresponding kinetic rates in the absence and presence of intact ribosome particles.
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Redox-modulated SNX25 as a novel regulator of GPCR-G protein signaling from endosomes
Redox Biology. 2024. Zhang, Yulong et al. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:
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Catalytic and noncatalytic functions of DNA polymerase $\kappa$ in translesion DNA synthesis
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2024. Sell{\'e}s-Baiget, Selene et al. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
ABSTRACT: Translesion DNA synthesis (TLS) is a cellular process that enables the bypass of DNA lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (Pol kappa) has the distinctive ability to bypass minor groove DNA adducts in vitro. However, Pol kappa is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base-editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Pol kappa during lesion bypass.
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Reliability of Serum-Derived Connectome Indicators in Identifying Cirrhosis
JOURNAL OF PROTEOME RESEARCH. 2024. Guo, Jisheng et al. Research Department, The sixth people’s hospital of Zhengzhou, Zhengzhou 450000, China
ABSTRACT: Patients with cirrhosis face a heightened risk of complications, underscoring the importance of identification. We have developed a Connectome strategy that combines metabolites with peptide spectral matching (PSM) in proteomics to integrate metabolomics and proteomics, identifying specific metabolites bound to blood proteins in cirrhosis using open search proteomics methods. Analysis methods including Partial Least Squares Discriminant Analysis (PLS-DA), Uniform Manifold Approximation and Projection (UMAP), and hierarchical clustering were used to distinguish significant differences among the Cirrhosis group, Chronic Hepatitis B (CHB) group, and Healthy group.
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Epigenetic therapy potentiates transposable element transcription to create tumor-enriched antigens in glioblastoma cells
NATURE GENETICS. 2024. Jang, H Josh et al. Department of Genetics, Washington University School of Medicine, St. Louis, MO, USA
ABSTRACT: Inhibiting epigenetic modulators can transcriptionally reactivate transposable elements (TEs). These TE transcripts often generate unique peptides that can serve as immunogenic antigens for immunotherapy. Here, we ask whether TEs activated by epigenetic therapy could appreciably increase the antigen repertoire in glioblastoma, an aggressive brain cancer with low mutation and neoantigen burden. We treated patient-derived primary glioblastoma stem cell lines, an astrocyte cell line and primary fibroblast cell lines with epigenetic drugs, and identified treatment-induced, TE-derived transcripts that are preferentially expressed in cancer cells.
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In situ chemoproteomic profiling reveals itaconate inhibits de novo purine biosynthesis in pathogens
CELL REPORTS. 2024. Liu, Zihua et al. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
ABSTRACT: Itaconate serves as an immune-specific metabolite that regulates gene transcription and metabolism in both host and pathogens. S-itaconation is a post-translational modification that regulates immune response; however, its antimicrobial mechanism under the physiological condition remains unclear. Here, we apply a bioorthogonal itaconate probe to perform global profiling of S-itaconation in living pathogens, including S. Typhimurium, S. aureus, , and P. aeruginosa. . Some functional enzymes are covalently modified by itaconate, including those involved in the de novo purine biosynthesis pathway.
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高分辨液相色谱质谱联用仪在 蛋白质化学修饰中的应用.
Experimental Technology & Management. 2024. 汪会玲 et al.
ABSTRACT:
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Dissecting diazirine photo-reaction mechanism for protein residue-specific cross-linking and distance mapping
NATURE COMMUNICATIONS. 2024. Jiang, Yida et al. Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
ABSTRACT: While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism.
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Venom variation among the three subspecies of the North African mountain viper Vipera monticola Saint Girons 1953
Biochimie. 2024. Damm, Maik et al. CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão, Universidade do Porto, 4485-661, Vairão, Portugal
ABSTRACT: The North African mountain viper (Vipera monticola) is a medically relevant venomous snake distributed in Morocco, Algeria, and Tunisia. Three subspecies of V.monticola, exhibiting differences in morphotypes and dietary regimes, are currently recognised: V.m. monticola, V.m. atlantica, and V.m. saintgironsi. Through the application of snake venomics, we analysed the venoms of specimens of Moroccan origin belonging to each of the three subspecies. Snake venom metalloproteinases (svMP), snake venom serine proteases (svSP), C-type lectin and C-type lectin-related proteins (CTL), and phospholipases A2 (PLA2) were predominant, with PLA2 being the most abundant toxin family overall.
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ZASP: A highly compatible and sensitive ZnCl2-precipitation assisted sample preparation method for proteomic analysis
Molecular & cellular proteomics : MCP. 2024. Shao, Xianfeng et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
ABSTRACT: Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT).
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Venomics and Peptidomics of Palearctic Vipers: A Clade-Wide Analysis of Seven Taxa of the Genera Vipera, Montivipera, Macrovipera, and Daboia across Türkiye
JOURNAL OF PROTEOME RESEARCH. 2024. Damm, Maik et al. Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
ABSTRACT: Snake venom variations are a crucial factor to understand the consequences of snakebite envenoming worldwide, and therefore it is important to know about toxin composition alterations between taxa. Palearctic vipers of the genera Vipera, Montivipera, Macrovipera, and Daboia have high medical impacts across the Old World. One hotspot for their occurrence and diversity is Turkiye, located on the border between continents, but many of their venoms remain still understudied. Here, we present the venom compositions of seven Turkish viper taxa.
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Identifying PE2 and PE5 Proteins from Existing Mass Spectrometry Data Using pFind
Journal of Proteome Research. 2024. Wei, Qianzhou et al. Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and MOE Key Laboratory of Tumor Molecular Biology, Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China
ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) aims to identify all proteins encoded by the human genome. Currently, the human proteome still contains approximately 2000 PE2-PE5 proteins, referring to annotated coding genes that lack sufficient protein-level evidence. During the past 10 years, it has been increasingly difficult to identify PE2-PE5 proteins in C-HPP approaches due to the limited occurrence. Therefore, we proposed that reanalyzing massive MS data sets in repository with newly developed algorithms may increase the occurrence of the peptides of these proteins.
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Redox-Modulated SNX25 as a Novel Regulator of GPCR-G Protein Signaling from Endosomes
Redox Biology. 2024. Zhang, Yulong et al. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:
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Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74
Frontiers in Immunology. 2024. Jiaru Song1 et al. Department of Pediatrics, Peking University First Hospital, Beijing, China
ABSTRACT:
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APIR: Aggregating Universal Proteomics Database Search Algorithms for Peptide Identification with FDR Control
Genomics, Proteomics & Bioinformatics. 2024. Chen, Yiling Elaine et al. Department of Statistics and Data Science, University of California
ABSTRACT:
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PIPI2: Sensitive Tag-Based Database Search to Identify Peptides with Multiple Post-translational Modifications
Journal of Proteome Research. 2024. Shengzhi Lai et al. Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Hong Kong, Hong Kong, China
ABSTRACT: Peptide identification is important in bottom-up proteomics. Post-translational modifications (PTMs) are crucial in regulating cellular activities. Many database search methods have been developed to identify peptides with PTMs and characterize the PTM patterns. However, the PTMs on peptides hinder the peptide identification rate and the PTM characterization precision, especially for peptides with multiple PTMs. To address this issue, we present a sensitive open search engine, PIPI2, with much better performance on peptides with multiple PTMs than other methods.
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Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids
Nature Communications. 2024. Liu, Dan-Dan et al. Life Sciences Institute, Department of Medical Oncology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310058, China
ABSTRACT: Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas.
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MS-PyCloud: A Cloud Computing-Based Pipeline for Proteomic and Glycoproteomic Data Analyses
Analytical Chemistry. 2024. Hu, Yingwei et al. Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21231, United States
ABSTRACT: Rapid development and wide adoption of mass spectrometry-based glycoproteomic technologies have empowered scientists to study proteins and protein glycosylation in complex samples on a large scale. This progress has also created unprecedented challenges for individual laboratories to store, manage, and analyze proteomic and glycoproteomic data, both in the cost for proprietary software and high-performance computing and in the long processing time that discourages on-the-fly changes of data processing settings required in explorative and discovery analysis.
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VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates
Nature Communications. 2024. Ann Schirin Mirsanaye et al. Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200, Copenhagen, Denmark
ABSTRACT: The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved alpha-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors.
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Research on the Upper Limit of Accuracy for Predicting Theoretical Tandem Mass Spectrometry
Journal of Computer and Communications. 2024. He, Changjiu et al. School of Computer Science and Technology, Shandong University of Technology
ABSTRACT:
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Stable isotope labeling-based two-step derivatization strategy for analysis of Phosphopeptides
Journal of Proteomics. 2024. Lunfei Zou et al. Key Laboratory of Optoelectronic Chemical Materials and Devices, Ministry of Education, School of Optoelectronic Materials & Technology, Jianghan University, Wuhan 430056, Hubei, People's Republic of China
ABSTRACT: Investigating site-specific protein phosphorylation remains a challenging task. The present study introduces a two-step chemical derivatization method for accurate identification of phosphopeptides. Methylamine neutralizes carboxyl groups, thus reducing the adsorption of non-phosphorylated peptides during enrichment, while dimethylamine offers a cost-effective reagent for stable isotope labeling of phosphorylation sites. The derivatization improves the mass spectra obtained through liquid chromatography-tandem mass spectrometry.
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Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides
. 2024. Humberto J. Ferreira et al. Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland
ABSTRACT: Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs.
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Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells
Nature Communications. 2024. Hao Hu et al. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
ABSTRACT: Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner.
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PgxSAVy: A tool for comprehensive evaluation of variant peptide quality in proteogenomics--catching the (un) usual suspects
Computational and Structural Biotechnology Journal. 2024. Anurag Raj  et al. G. N. Ramachandran Knowledge Centre for Genomics Informatics, CSIR – Institute of Genomics and Integrative Biology, New Delhi, India
ABSTRACT: Variant peptides resulting from single nucleotide polymorphisms (SNPs) can lead to aberrant protein functions and have translational potential for disease diagnosis and personalized therapy. Variant peptides detected by proteogenomics are fraught with high number of false positives, but there is no uniform and comprehensive approach to assess variant quality across analysis pipelines. Despite class -specific FDR along with ad -hoc filters, the problem is far from solved. These protocols are typically manual and tedious, and thus not uniform across labs.
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CloudProteoAnalyzer: scalable processing of big data from proteomics using cloud computing
Bioinformatics Advances. 2024. Li, Jiancheng et al. Department of Computer Science and Engineering, University of North Texas
ABSTRACT: Summary: Shotgun proteomics is widely used in many system biology studies to determine the global protein expression profiles of tissues, cultures, and microbiomes. Many non-distributed computer algorithms have been developed for users to process proteomics data on their local computers. However, the amount of data acquired in a typical proteomics study has grown rapidly in recent years, owing to the increasing throughput of mass spectrometry and the expanding scale of study designs. This presents a big data challenge for researchers to process proteomics data in a timely manner.
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Apoptosis releases hydrogen sulfide to inhibit Th17 cell differentiation
Cell Metabolism. 2024. Qianmin Ou et al. Key Laboratory of Stem Cells and Tissue Engineering (Sun Yat-Sen University), Ministry of Education, Guangzhou 510080, China
ABSTRACT: Over 50 billion cells undergo apoptosis each day in an adult human to maintain immune homeostasis. Hydrogen sulfide (H2S) is also required to safeguard the function of immune response. However, it is unknown whether apoptosis regulates H2S production. Here, we show that apoptosis-deficient MRL/lpr (B6.MRL-Faslpr/J) and Bim-/- (B6.129S1-Bcl2l11tm1.1Ast/J) mice exhibit significantly reduced H2S levels along with aberrant differentiation of Th17 cells, which can be rescued by the additional H2S. Moreover, apoptotic cells and vesicles (apoVs) express key H2S-generating enzymes and generate a significant amount of H2S, indicating that apoptotic metabolism is an important source of H2S.
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The Deconvolution Method for Obtaining Correspondence in Data-Independent Acquisition Mass Spectrometry Data
. 2024. Lyu, Mingming et al. College of Computer Science and Technology, Shandong University of Technology, Zibo, China
ABSTRACT:
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Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells
Nature communications. 2024. Zhao, Lili et al. CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, State Key Laboratory of Medical Proteomics, Dalian
ABSTRACT: Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells.
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Docking a flexible basket onto the core of the nuclear pore complex
NATURE CELL BIOLOGY. 2024. Stankunas, Edvinas et al. Max Perutz Labs, Vienna Biocenter Campus, University of Vienna and Medical University of Vienna, Vienna, Austria
ABSTRACT: The nuclear basket attaches to the nucleoplasmic side of the nuclear pore complex (NPC), coupling transcription to mRNA quality control and export. The basket expands the functional repertoire of a subset of NPCs in Saccharomyces cerevisiae by drawing a unique RNA/protein interactome. Yet, how the basket docks onto the NPC core remains unknown. By integrating AlphaFold-based interaction screens, electron microscopy and membrane-templated reconstitution, we uncovered a membrane-anchored tripartite junction between basket and NPC core.
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Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2024. Novikova, Irina V et al. Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States
ABSTRACT: The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O-2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction.
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An Optimized Miniaturized Filter-Aided Sample Preparation Method for Sensitive Cross-Linking Mass Spectrometry Analysis of Microscale Samples
ANALYTICAL CHEMISTRY. 2024. He, Yu et al. Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, China
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples.
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Full-length GSDME mediates pyroptosis independent from cleavage
NATURE CELL BIOLOGY. 2024. Zhou, Bo et al. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China
ABSTRACT: Gasdermin (GSDM) family proteins, known as the executors of pyroptosis, undergo protease-mediated cleavage before inducing pyroptosis. We here discovered a form of pyroptosis mediated by full-length (FL) GSDME without proteolytic cleavage. Intense ultraviolet-C irradiation-triggered DNA damage activates nuclear PARP1, leading to extensive formation of poly(ADP-ribose) (PAR) polymers. These PAR polymers are released to the cytoplasm, where they activate PARP5 to facilitate GSDME PARylation, resulting in a conformational change in GSDME that relieves autoinhibition.
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An IDR-dependent mechanism for nuclear receptor control of Mediator interaction with RNA polymerase II
MOLECULAR CELL. 2024. Zhao, Haiyan et al. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical School, Aurora, CO 80045, USA
ABSTRACT: The essential Mediator (MED) coactivator complex plays a well-understood role in regulation of basal transcription in all eukaryotes, but the mechanism underlying its role in activator-dependent transcription remains unknown. We investigated modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) by antagonistic effects of the MED26 subunit and the CDK8 kinase module (CKM). Biochemical analysis of CKM-MED showed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction.
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Calcium modulates the tethering of BCOR-PRC1. 1 enzymatic core to KDM2B via liquid-liquid phase separation
COMMUNICATIONS BIOLOGY. 2024. Chen, Rui et al. State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT: Recruitment of non-canonical BCOR-PRC1.1 to non-methylated CpG islands via KDM2B plays a fundamental role in transcription control during developmental processes and cancer progression. However, the mechanism is still largely unknown on how this recruitment is regulated. Here, we unveiled the importance of the Poly-D/E regions within the linker of BCOR for its binding to KDM2B. Interestingly, we also demonstrated that these negatively charged Poly-D/E regions on BCOR play autoinhibitory roles in liquid-liquid phase separation (LLPS) of BCORANK-linker-PUFD/PCGF1RAWUL.
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Structural insights into the human NuA4/TIP60 acetyltransferase and chromatin remodeling complex
SCIENCE. 2024. Yang, Zhenlin et al. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.
ABSTRACT: The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module.
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Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B
Elife. 2024. Daniel Ballmer et al. Univ Oxford, Dept Biochem, Oxford, England; Inst Cell Biol, Wellcome Ctr Cell Biol, Sch Biol Sci, Edinburgh, Scotland
ABSTRACT: The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin.
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Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis
Proceedings of the National Academy of Sciences. 2024. Menghan Mei  et al. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
ABSTRACT: The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm.
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The crosslinking sites and molecular conformation of gelatin hydrogel modified by transglutaminase
Food Hydrocolloids. 2024. Yunke Yang et al. College of Food Science, Southwest University, Chongqing, 400715, PR China
ABSTRACT: This work aimed to investigate the covalent crosslinking sites induced by transglutaminase (TGase, EC 2.3.2.13) in gelatin hydrogel and their impact on the gelatin molecular conformation. LC-MS/MS results demonstrated that Lys644 and Gln1351 of alpha 1 chain and Gln19 of alpha 2 chain were the main sites. The results of molecular dynamic (MD) simulation indicated that at the moderate degree of covalent crosslinking (13.55%), Radius of gyration (Rg) values decreased from 11.5 to 10.0 nm at 4 degrees C and led to a more compact structure, therefore enhancing structural tightness and gel strength.
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On the utility of the extracted ion chromatograms for assigning the conjugation sites and side reactions in bioconjugates synthesized by the maleimide-thiol chemistry
Microchemical Journal. 2024. Satomy Pousa et al. Department of Proteomics, Mass Spectrometry Laboratory, Center for Genetic Engineering and Biotechnology, Avenida 31 e/ 158 y 190, Cubanacán, Playa, PO. Box 6162, La Habana, Cuba
ABSTRACT:
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Structure of the plant plastid-encoded RNA polymerase
Cell. 2024. Á Vergara-Cruces et al. Department of Biochemistry and Metabolism, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
ABSTRACT: Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba).
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Cryo-EM structures of the plant plastid-encoded RNA polymerase
Cell. 2024. XX Wu et al. Key Laboratory of Synthetic Biology, Key Laboratory of Plant Design, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China
ABSTRACT: Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions.
[more...]
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TopBP1 utilises a bipartite GINS binding mode to support genome replication
Nature communications. 2024. M Day et al. Molecular Genetics II, Center of Medical Biotechnology, University of Duisburg-Essen, Universitätsstraße 2-5, 45141, Essen, Germany
ABSTRACT: Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1.
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Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins
EMBO Journal. 2024. Zhenwei Zhang et al. Cellular BiochemistryMax-Planck-Institute for Multidisciplinary Sciences Am Fassberg 11 37077 Göttingen Germany
ABSTRACT:
Use: pLink



Structural insights into histone exchange by human SRCAP complex
CELL DISCOVERY. 2024. J Yu et al. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, New Cornerstone Science Laboratory, State Key Laboratory of Genetic Engineering and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai, China
ABSTRACT: Histone variant H2A.Z is found at promoters and regulates transcription. The ATP-dependent chromatin remodeler SRCAP complex (SRCAP-C) promotes the replacement of canonical histone H2A-H2B dimer with H2A.Z-H2B dimer. Here, we determined structures of human SRCAP-C bound to H2A-containing nucleosome at near-atomic resolution. The SRCAP subunit integrates a 6-subunit actin-related protein (ARP) module and an ATPase-containing motor module. The ATPase-associated ARP module encircles half of the nucleosome along the DNA and may restrain net DNA translocation, a unique feature of SRCAP-C.
[more...]
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An extensive disulfide bond network prevents tail contraction in Agrobacteriumtumefaciens phage Milano
. 2024. RR Sonani et al. Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, 22903, USA
ABSTRACT:
Use: pLink



Multi-scale structures of the mammalian radial spoke and divergence of axonemal complexes in ependymal cilia
Nature Communications. 2024. Meng, Xueming et al. Key Laboratory of RNA Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, 200031, China
ABSTRACT: Radial spokes (RS) transmit mechanochemical signals between the central pair (CP) and axonemal dynein arms to coordinate ciliary motility. Atomic-resolution structures of metazoan RS and structures of axonemal complexes in ependymal cilia, whose rhythmic beating drives the circulation of cerebrospinal fluid, however, remain obscure. Here, we present near-atomic resolution cryo-EM structures of mouse RS head-neck complex in both monomer and dimer forms and reveal the intrinsic flexibility of the dimer.
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Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1
Nature Structural & Molecular Biology. 2024. D{\"u}ring, Jonas et al. Research Group ‘Ubiquitin Signaling Specificity’, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany
ABSTRACT: Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry.
[more...]
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RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis
Cell Reports. 2024. Prusty et al. Department of Biochemistry 1, Biocenter, University of Würzburg, 97074 Würzburg, Germany
ABSTRACT: RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export.
[more...]
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Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair
Nature Structural & Molecular Biology. 2024. Kokic, Goran et al. Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
ABSTRACT: During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation.
[more...]
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A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass
Molecular Cell. 2024. Yuqiu Sun et al. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing 102206, China
ABSTRACT:
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Precision N-glycoproteomics reveals the essential role of the extracellular matrix in tropomyosin allergy in a mouse model
Food Science and Human Wellness. 2024. Liu, Guirong et al. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R.China;
ABSTRACT:
Use: pGlyco



Expanding N-glycopeptide identifications by modeling fragmentation, elution, and glycome connectivity
NATURE COMMUNICATIONS. 2024. Klein, Joshua et al. Program for Bioinformatics, Boston University, Boston, MA, US
ABSTRACT: Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a dynamically adapting relative retention time model for detecting and correcting ambiguous glycan assignments that are difficult to detect from fragmentation alone, a layered approach to glycopeptide fragmentation modeling that improves N-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further.
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High-abundance serum glycoproteins as valuable resources for glycopeptide standards
CARBOHYDRATE POLYMERS. 2024. Li, Jun et al. Laboratory for Disease Glycoproteomics, College of Life Sciences, Northwest University, Xi'an 710069, PR China
ABSTRACT: High-abundance serum proteins, mostly modified by N-glycans, are usually depleted from human sera to achieve in-depth analyses of serum proteome and sub-proteomes. In this study, we show that these high-abundance glycoproteins (HAGPs) can be used as valuable standard glycopeptide resources, as long as the structural features of their glycans have been well defined at the glycosite-specific level. By directly analyzing intact glycopeptides enriched from serum, we identified 1322 unique glycopeptides at 48 N-glycosites from the top 12 HAGPs (19 subclasses).
[more...]
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Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow
Cell Reports Methods. 2024. Taewook Kang et al. State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, 200031, Shanghai, China
ABSTRACT:
Use: pGlyco



ppmFixer: a mass error adjustment for pGlyco3. 0 to correct near-isobaric mismatches
Glycobiology. 2024. Adams, Trevor M et al. Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens 30602, Georgia
ABSTRACT: Modern glycoproteomics experiments require the use of search engines due to the generation of countless spectra. While these tools are valuable, manual validation of search engine results is often required for detailed analysis of glycopeptides as false-discovery rates are often not reliable for glycopeptide data. Near-isobaric mismatches are a common source of misidentifications for the popular glycopeptide-focused search engine pGlyco3.0, and in this technical note we share a strategy and script that improves the accuracy of the search utilizing two manually validated datasets of the glycoproteins CD16a and HIV-1 Env as proof-of-principle.
Use: pGlyco




2023




Comparative analysis of chemical cross-linking mass spectrometry data indicates that protein STY residues rarely react with N-hydroxysuccinimide ester cross-linkers
Journal of Proteome Research. 2023. Cao, Yong et al. National Institute of Biological Sciences, Beijing, Beijing 102206, China
ABSTRACT:
Use: pChem; pFind; pLink



Dynamic localization of the chromosomal passenger complex is controlled by the orphan kinesins KIN-A and KIN-B in the kinetoplastid parasite Trypanosoma brucei
eLife. 2023. Ballmer, Daniel et al. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, The Wellcome Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, Edinburgh, EH9 3BF, United Kingdom
ABSTRACT:
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Cross-linking mass spectrometry uncovers interactions between high-density lipoproteins and the SARS-CoV-2 spike glycoprotein
Molecular & cellular proteomics : MCP. 2023. Burnap, Sean A et al. College of Chemistry, Jilin University, Changchun, 130012, Jilin, China
ABSTRACT: High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein.
[more...]
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Cross-linking Mass Spectrometry Uncovers Interactions Between High-density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein
Molecular & cellular proteomics : MCP. 2023. Burnap, Sean A et al. College of Chemistry, Jilin University, Changchun, 130012, Jilin, China
ABSTRACT: High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein.
[more...]
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TopBP1 utilises a bipartite GINS binding mode to activate the replicative helicase
J Biol Chem. 2023. Day, Matthew et al. Department of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Göttingen, Germany
ABSTRACT:
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Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1
Molecular cell. 2023. Abini-Agbomson, Stephen et al. Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
ABSTRACT: The intricate regulation of chromatin plays a key role in controlling genome architecture and accessibility. Histone lysine methyltransferases regulate chromatin by catalyzing the methylation of specific histone residues but are also hypothesized to have equally important non-catalytic roles. SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation, and is dysregulated in several cancers. Many of these processes were linked to its catalytic activity.
[more...]
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A tyrosine, histidine-selective bifunctional cross-linker for protein structure analysis
Talanta. 2023. Yan, Qibo et al. Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
ABSTRACT: Chemical cross-linking mass spectrometry (XL-MS) significantly contributes to the analysis of protein structures and the elucidation of protein-protein interactions. Currently available cross-linkers mainly target N-terminus, lysine, glutamate, aspartate, and cysteine residues in protein. Herein, a bifunctional cross-linker, named [4,4'-(disulfanediylbis(ethane-2,1-diyl)) bis(1-methyl-1,2,4-triazolidine-3,5-dione)] (DBMT) has been designed and characterized aiming to extremely expand the application of XL-MS approach.
[more...]
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Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein
The Journal of biological chemistry. 2023. P{\"o}rschke, Marius et al. Department of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Göttingen, Germany
ABSTRACT: The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm.
[more...]
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Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel
International Journal of Biological Macromolecules. 2023. Jagadeeshaprasad, Mashanipalya G et al. Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
ABSTRACT: VAR2CSA, a multidomain Plasmodium falciparum protein, mediates the adherence of parasite-infected red blood cells to chondroitin 4-sulfate (C4S) in the placenta, contributing to placental malaria. Therefore, detailed understanding of VAR2CSA structure likely help developing strategies to treat placental malaria. The VAR2CSA ectodomain consists of an N-terminal segment (NTS), six Duffy binding-like (DBL) domains, and three interdomains (IDs) present in sequence NTS-DBL1x-ID1-DBL2x-ID2-DBL3x-DBL4epsilon-ID3-DBL5epsilon-DBL6epsilon.
[more...]
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Fast cross-linking by DOPA2 promotes the capturing of a stereospecific protein complex over nonspecific encounter complexes
Biophysics Reports. 2023. Jian-Hua, Wang et al. National Institute of Biological Sciences (NIBS), Beijing 102206, China
ABSTRACT: Transient and weak protein-protein interactions are essential to many biochemical reactions, yet are technically challenging to study. Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) provides a powerful tool in the analysis of such interactions. Central to this technology are chemical cross-linkers. Here, using two transient heterodimeric complexes EIN/HPr and EIIAGlc/EIIBGlc as our model systems, we evaluated the effects of two amine-specific homo-bifunctional cross-linkers with different reactivities.
[more...]
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Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells
Redox Biology. 2023. Wu, Ting et al. Center for Chemical Biology and Drug Discovery, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT: Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation.
[more...]
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Simultaneous enrichment and profiling of intact N-linked, O-GalNAc, and O-GlcNAcylated glycopeptides
Angewandte Chemie International Edition. 2023. Liu, Jialin et al. College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking-Tsinghua Center for Life Sciences, Synthetic and Functional Biomolecules Center, and Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871 China
ABSTRACT:
Use: pFind; pDeep; pGlyco



Threatened North African seagrass meadows have supported green turtles for millennia
Proceedings of the National Academy of Sciences. 2023. de Kock, Willemien et al. Groningen Institute of Archaeology, Faculty of Arts, University of Groningen, 9712 ER Groningen, Netherlands
ABSTRACT:
Use: pFind



Cyanobacterial phycobilisome allostery as revealed by quantitative mass spectrometry
Biochemistry. 2023. liuhaijun et al. Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States
ABSTRACT: Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod-core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment-protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane.
[more...]
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ReCom: A semi-supervised approach to ultra-tolerant database search for improved identification of modified peptides
Journal of Proteomics. 2023. Jesús Vázquez et al. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid 28029, Spain
ABSTRACT: Open-search methods allow unbiased, high-throughput identification of post-translational modifications in proteins at an unprecedented scale. The performance of current open-search algorithms is diminished by experimental errors in the determination of the precursor peptide mass. In this work we propose a semisupervised open search approach, called ReCom, that minimizes this effect by taking advantage of a priori known information from a reference database, such as Unimod or a database provided by the user.
[more...]
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Sulfoproteomics workflow with precursor ion accurate mass shift analysis reveals novel tyrosine sulfoproteins in the golgi
Journal of Proteome Research. 2023. Kweon, Hye Kyong et al. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1085, United States
ABSTRACT: Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search.
[more...]
Use: pFind



Combining tags of various lengths benefits peptide identification in bottom-up proteomics
. 2023. Lai, Shengzhi et al. Department of Electronic and Computer Engineering
ABSTRACT:
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DbyDeep: exploration of MS-detectable peptides via deep learning
Analytical Chemistry. 2023. Juho Son et al. Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea; Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea
ABSTRACT: Predicting peptide detectability is useful in a varietyof massspectrometry (MS)-based proteomics applications, particularly targetedproteomics. However, most machine learning-based computational methodshave relied solely on information from the peptide itself, such asits amino acid sequences or physicochemical properties, despite thefact that peptides detected by MS are dependent on many factors, includingprotein sample preparation, digestion, separation, ionization, andprecursor selection during MS experiments.
[more...]
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Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS
Journal of Assisted Reproduction and Genetics. 2023. Yuqin Yang et al. Department of Reproductive Medicine, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, China
ABSTRACT:
Use: pFind



Modulating Liquid--Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection
ACS Central Science. 2023. Liu Min et al. Department of Chemistry and Center for Cell & Developmental Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
ABSTRACT: Signaling proteins often form biomolecular condensates through liquid-liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott-Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex.
[more...]
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Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of fission yeast wtf meiotic drivers
Nature Communications. 2023. Jin-Xin Zheng et al. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, 102206, China
ABSTRACT: Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. In several fission yeast species, single-gene KMDs belonging to the wtf gene family exert selfish killing by expressing a toxin and an antidote through alternative transcription initiation. Here we investigate how the toxin and antidote products of a wtf-family KMD gene can act antagonistically.
[more...]
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Proteome landscapes of human hepatocellular carcinoma and intrahepatic cholangiocarcinoma
Molecular & cellular proteomics : MCP. 2023. Xiao Yi et al. Center for ProtTalks, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China
ABSTRACT: Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types.
[more...]
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Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi
Journal of Proteome Research. 2023. Kweon, Hye Kyong et al. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1085, United States
ABSTRACT: Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search.
[more...]
Use: pFind



Combining Tags of Various Lengths Benefits Peptide Identification in Bottom-up Proteomics
. 2023. Lai, Shengzhi et al. Department of Electronic and Computer Engineering
ABSTRACT:
Use: pFind



O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins
Nature Cell Biology. 2023. Yulin Chen et al. Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China
ABSTRACT: N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins.
[more...]
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Disruption of the productive encounter complex results in dysregulation of DIAPH1 activity
Journal of Biological Chemistry. 2023. Gregory G. Theophall et al. Max Planck Inst Multidisciplinary Sci, D-37077 Gottingen, Germany
ABSTRACT: The diaphanous-related formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia, and cardiovascular diseases.
[more...]
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Current perspectives on mass spectrometry-based immunopeptidomics: the computational angle to tumor antigen discovery
. 2023. Bing Zhang et al. Ludwig Institute for Cancer Research, University of Lausanne, Lausanne, Switzerland
ABSTRACT:
Use: pFind; pDeep



Histones with an unconventional DNA-binding mode in vitro are major chromatin constituents in the bacterium Bdellovibrio bacteriovorus
Nature Microbiology. 2023. Antoine Hocher et al. Antoine Hocher, Karolin Luger, Tobias Warnecke. Medical Research Council London Institute of Medical Sciences, London, UK Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK
ABSTRACT: Histone proteins bind DNA and organize the genomes of eukaryotes and most archaea, whereas bacteria rely on different nucleoid-associated proteins. Homology searches have detected putative histone-fold domains in a few bacteria, but whether these function like archaeal/eukaryotic histones is unknown. Here we report that histones are major chromatin components in the bacteria Bdellovibrio bacteriovorus and Leptospira interrogans. Patterns of sequence evolution suggest important roles for histones in additional bacterial clades.
[more...]
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Ubiquitination-mediated Golgi-to-endosome sorting determines the toxin-antidote duality of wtf meiotic drivers
. 2023. Jin-Xin Zheng et al. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University,
ABSTRACT:
Use: pFind



Grouping groupers in the Mediterranean: Ecological baselines revealed by ancient proteins
Ecology and Evolution. 2023. Rachel M. Winter et al. Groningen Institute of Archaeology, University of Groningen, Groningen, The Netherlands
ABSTRACT: Marine historical ecology provides a means to establish baselines to inform current fisheries management. Groupers (Epinephelidae) are key species for fisheries in the Mediterranean, which have been heavily overfished. Species abundance and distribution prior to the 20th century in the Mediterranean remains poorly known. To reconstruct the past biogeography of Mediterranean groupers, we investigated whether Zooarchaeology by Mass Spectrometry (ZooMS) can be used for identifying intra-genus grouper bones to species level.
[more...]
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Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis
The Journal of Clinical Investigation. 2023. Korawit Kanjana et al. dmitry.temiakov@jefferson.edu
ABSTRACT: BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Valpha1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins.
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Proteogenomic analysis reveals RNA as a source for tumor-agnostic neoantigen identification
Nature Communications. 2023. Celina Tretter et al. TUM
ABSTRACT: RNA variants derived from cancer-associated RNA editing events can be a source of neoantigens. Here, based on a proteogenomic pipeline combining DNA and RNA sequencing with MS-based immunopeptidomics, the authors identity and validate potential neoantigen candidates in patients with different tumor entities, highlighting RNA as important neoantigen source.Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens.
[more...]
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MetaPep: A core peptide database for faster human gut metaproteomics database searches
Computational and Structural Biotechnology Journal. 2023. Sun, Zhongzhi et al. School of Pharmaceutical Sciences, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
ABSTRACT: Metaproteomics has increasingly been applied to study functional changes in the human gut microbiome. Peptide identification is an important step in metaproteomics research, with sequence database search (SDS) and spectral library search (SLS) as the two main methods to identify peptides. However, the large search space in metaproteomics studies causes significant challenges for both identification methods. Moreover, with the development of mass spectrometry, it is now feasible to perform metaproteomic projects involving 100-1000 individual microbiomes.
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Sijunzi decoction ameliorates gastric precancerous lesions via regulating oxidative phosphorylation based on proteomics and metabolomics
Journal of Ethnopharmacology. 2023. Zhu, Yanning et al. School of Pharmacy, Lanzhou University, Lanzhou, PR China
ABSTRACT: ETHNOPHARMACOLOGICAL RELEVANCE: Sijunzi decoction (SJZD), a traditional Chinese medicine formula, is commonly used in clinical practice for the treatment of gastric precancerous lesions (GPL). However, the mechanism of gastric protection is not fully understood.AIMS OF THE STUDY: The purpose of this study was to systematically evaluate the efficacy of SJZD in blocking the development of GPL and to reveal the underlying mechanism.METHODS: First, we established a rat model of GPL, which was induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) combined with an irregular diet and 40% ethanol.
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Deep learning prediction boosts phosphoproteomics-based discoveries through improved phosphopeptide identification
bioRxiv. 2023. Yi, Xinpei et al. Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas, USA
ABSTRACT: Shotgun phosphoproteomics enables high-throughput analysis of phosphopeptides in biological samples, but low phosphopeptide identification rate in data analysis limits the potential of this technology. Here we present DeepRescore2, a computational workflow that leverages deep learning-based retention time and fragment ion intensity predictions to improve phosphopeptide identification and phosphosite localization. Using a state-of-the-art computational workflow as a benchmark, DeepRescore2 increases the number of correctly identified peptide-spectrum matches by 17% in a synthetic dataset and identifies 19%-46% more phosphopeptides in biological datasets.
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Threatened North African seagrass meadows have supported green turtle populations for millennia
Proceedings of the National Academy of Sciences. 2023. de Kock, Willemien et al. Groningen Institute of Archaeology, Faculty of Arts, University of Groningen, 9712 ER Groningen, Netherlands
ABSTRACT: "Protect and restore ecosystems and biodiversity" is the second official aim of the current UN Ocean Decade (2021 to 2030) calling for the identification and protection of critical marine habitats. However, data to inform policy are often lacking altogether or confined to recent times, preventing the establishment of long-term baselines. The unique insights gained from combining bioarchaeology (palaeoproteomics, stable isotope analysis) with contemporary data (from satellite tracking) identified habitats which sea turtles have been using in the Eastern Mediterranean over five millennia.
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Conformational cycle of human polyamine transporter ATP13A2
Nature Communications. 2023. Jianqiang Mu et al. So Univ Sci & Technol, Sch Life Sci, Dept Immunol & MicroBiol, Shenzhen 518055, Guangdong, Peoples R China; Int Campus Zhejiang Univ, Prov Int Sci & Technol Cooperat Base Engn Biol, Haining 314400, Peoples R China; Zhejiang Univ, Shanghai Inst Adv Study, Inst Quantitat Biol, Coll Life Sci, Hangzhou 310027, Peoples R China
ABSTRACT: ATP13A2 is a lysosomal polyamine transporter, mutated in several diseases including juvenile-onset Parkinson's disease. Here, the authors report structures of human ATP13A2 in six distinct intermediate states, illustrating most of its conformational cycle.Dysregulation of polyamine homeostasis strongly associates with human diseases. ATP13A2, which is mutated in juvenile-onset Parkinson's disease and autosomal recessive spastic paraplegia 78, is a transporter with a critical role in balancing the polyamine concentration between the lysosome and the cytosol.
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Molecular characterization of extracellular vesicles derived from follicular fluid of women with and without PCOS: integrating analysis of differential miRNAs and proteins reveals vital molecules involving in PCOS
Journal of Assisted Reproduction and Genetics. 2023. Yang, Yuqin et al. Nanjing Med Univ, Womens Hosp, Nanjing Matern & Child Hlth Care Hosp, Dept Reprod Med, Nanjing, Peoples R China
ABSTRACT: PurposeTo elucidate the characterization of extracellular vesicles (EVs) in the follicular fluid-derived extracellular vesicles (FF-EVs) and discover critical molecules and signaling pathways associating with the etiology and pathobiology of PCOS, the differentially expressed miRNAs (DEmiRNAs) and differentially expressed proteins profiles (DEPs) were initially explored and combinedly analyzed.MethodsFirst, the miRNA and protein expression profiles of FF-EVs in PCOS patients and control patients were compared by RNA-sequencing and tandem mass tagging (TMT) proteomic methods.
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Fully integrated on-line strategy for highly sensitive proteome profiling of 10–500 mammalian cells
Analyst. 2023. Yang, Yun et al. Chinese Acad Sci, Inst Neurosci, CAS Ctr Excellence Brain Sci & Intelligence Techno, State Key Lab Neurosci, Shanghai 200031, Peoples R China; Hong Kong Univ Sci &Technol, Dept Chem & Biol Engn, Clear Water Bay, Kowloon, Hong Kong, Peoples R China; Southern Univ Sci & Technol, Sch Sci, Dept Chem, 1088 Xueyuan Ave, Shenzhen 518055, Peoples R China
ABSTRACT: Recent development in proteomic sample preparation using nanofluidic devices has made single-cell proteome profiling possible. However, these nanofluidic devices require special expertise and costly nanopipetting instruments. They are also specially designed for single cells, are not well-suited for profiling rare samples consisting of a few hundred mammalian cells, arguably a more common need that remains a great challenge. Herein, we developed an easy-to-use and scalable device for processing low-input samples, which combined the merits of previously reported rare cell proteomic reactor (RCPR) and mixed-mode simple and integrated spintip-based proteomics technology, as an alternative to nanofluidic devices.
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VCF1 is an unconventional p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates
. 2023. Mirsanaye, Ann Schirin et al. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200, Copenhagen, Denmark
ABSTRACT:
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DbyDeep: Exploration of MS-Detectable Peptides via Deep Learning
Analytical Chemistry. 2023. Juho Son et al. Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea; Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea
ABSTRACT: Predicting peptide detectability is useful in a varietyof massspectrometry (MS)-based proteomics applications, particularly targetedproteomics. However, most machine learning-based computational methodshave relied solely on information from the peptide itself, such asits amino acid sequences or physicochemical properties, despite thefact that peptides detected by MS are dependent on many factors, includingprotein sample preparation, digestion, separation, ionization, andprecursor selection during MS experiments.
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Novel Proteoform Discovery by Precise Semi-De Novo Sequencing of Novel Junction Peptides
Analytical Chemistry. 2023. Zhitai Hao et al. Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Med Res Ctr, Beijing 100730, Peoples R China; Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ Hlth Sci Ctr, Ctr Precis Med Multi Res, Beijing 100191, Peoples R China
ABSTRACT: Alternative splicing allows a smallnumber of human genesto encodelarge amounts of proteoforms that play essential roles in normal anddisease physiology. Some low-abundance proteoforms may remain undiscovereddue to limited detection and analysis capabilities. Peptides coencodedby novel exons and annotated exons separated by introns are callednovel junction peptides, which are the key to identifying novel proteoforms.Traditional de novo sequencing does not take intoaccount the specificity in the composition of the novel junction peptideand is therefore not as accurate.
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Proteome Landscapes of Human Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma
Molecular & cellular proteomics : MCP. 2023. Xiao Yi et al. Center for ProtTalks, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China
ABSTRACT: Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types.
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RNA polymerase drives ribonucleotide excision DNA repair in E. coli
Cell. 2023. Nudler, Evgeny et al. New York Univ, Dept Biochem & Mol Pharmacol, Grossman Sch Med, New York, NY 10016 USA; New York Univ, Howard Hughes Med Inst, Grossman Sch Med, New York, NY 10016 USA
ABSTRACT: Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside mono -phosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull -downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy struc-tures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show spe-cific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states.
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Characterization of the natural peptidome of four leeches by integrated proteogenomics and pseudotargeted peptidomics
Analytical and Bioanalytical Chemistry. 2023. Jingmei Liao1 et al. Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai Res Ctr Modernizat Tradit Chinese Med, Natl Engn Res Ctr TCM Standardizat Technol, Shanghai 201203, Peoples R China; Nanjing Univ Chinese Med, Sch Chinese Mat Med, Nanjing 210023, Jiangsu, Peoples R China; Univ Chinese Acad Sci, 19A Yuquan Rd, Beijing 100049, Peoples R China
ABSTRACT: Animal-derived drugs are an indispensable part of folk medicine worldwide. However, their chemical constituents are poorly approached, which leads to the low level of the quality standard system of animal-derived drugs and further causes a chaotic market. Natural peptides are ubiquitous throughout the organism, especially in animal-derived drugs. Thus, in this study, we used multi-source leeches, including Hirudo nipponica (HN), Whitmania pigra (WP), Whitmania acranulata (WA), and Poecilobdella manillensis (PM), as a model.
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Cyanobacterial Phycobilisome Allostery as Revealed by Quantitative Mass Spectrometry
Biochemistry. 2023. liuhaijun et al. Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States
ABSTRACT: Phycobilisomes (PBSs) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. PBS, a multisubunit protein complex, has two major interfaces that comprise intrinsically disordered regions (IDRs): rod-core and core-membrane. IDRs do not form regular, three-dimensional structures on their own. Their presence in the photosynthetic pigment-protein complexes portends their structural and functional importance. A recent model suggests that PB-loop, an IDR located on the PBS subunit ApcE and C-terminal extension (CTE) of the PBS subunit ApcG, forms a structural protrusion on the PBS core-membrane side, facing the thylakoid membrane.
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Large-Scale Profiling of Unexpected Tryptic Cleaved Sites at Ubiquitinated Lysines
Journal of Proteome Research. 2023. Sun, Zhen et al. Anhui Med Univ, Hefei 230032, Peoples R China; Chinese Acad Med Sci, Natl Ctr Prot Sci Beijing, Res Unit Prote Res & Dev New Drug, State Key Lab Prote,Beijing Proteome Res Ctr,Inst, Beijing 102206, Peoples R China; Wuhan Univ, Sch Basic Med Sci, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery,Mi, Wuhan 430071, Peoples R China; Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Divers, Baoding 071002, Hebei, Peoples R China
ABSTRACT: Trypsin specifically cleaves the C-terminus of lysine and arginine residues but often fails to cleave modified lysines, such as ubiquitination, therefore resulting in the uncleaved K-e-GG peptides. Therefore, the cleaved ubiquitinated peptide identification was often regarded as false positives and discarded. Interestingly, unexpected cleavage at the K48-linked ubiquitin chain has been reported, suggesting the latent ability of trypsin to cleave ubiquitinated lysine residues. However, it remains unclear whether other trypsin-cleavable ubiquitinated sites are present.
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Linking chromatin acylation mark-defined proteome and genome in living cells
Cell. 2023. Qin, Fangfei et al. Shenzhen Bay Lab, Shenzhen 518055, Peoples R China; Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, Sch Life Sci, Key Lab Cell Proliferat & Differentiat, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn, Minist Educ, Beijing 100871, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Beijing 100871, Peoples R China
ABSTRACT: A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and b-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions.
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SET7 methylates the deubiquitinase OTUB1 at Lys 122 to impair its binding to E2 enzyme UBC13 and relieve its suppressive role on ferroptosis
The Journal of biological chemistry. 2023. Deng, Hongyan et al. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, P. R. China
ABSTRACT: The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122.
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Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis
Journal of Proteome Research. 2023. Gao, Jing et al. Analytical Research Center for Organic and Biological Molecules, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Media, Chinese Academy of Sciences, Shanghai, 201203, P. R. China
ABSTRACT: The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other.
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Mass spectrometry and spectroscopic characterization of a tetrameric photosystem I supercomplex from Leptolyngbya ohadii, a desiccation-tolerant cyanobacterium
Biochimica et biophysica acta. Bioenergetics. 2023. Niedzwiedzki, Dariusz M et al. Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA
ABSTRACT: Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy.
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Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics
The Plant Cell. 2023. Kim, Tae-Wuk et al. Carnegie Inst Sci, Dept Plant Biol, Stanford, CA 94305 USA
ABSTRACT: Combining TurboID-mediated proximity labeling with quantitative phosphoproteomics identifies BIN2 signaling components including kinase substrates in vivo, revealing cellular functions of BIN2.Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases.
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Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen
Molecular Immunology. 2023. Wang, Ye et al. Chinese Acad Med Sci, Peking Union Med Coll Hosp, Peking Union Med Coll, Dept Allergy, 1 Shuaifuyuan, Wangfujing 100730, Beijing, Peoples R China; Affiliated Canc Hosp Nanjing Med Univ, Jiangsu Canc Hosp, Jiangsu Inst Canc Res, Dept Pharm, 42,Baiziting Rd, Nanjing 210029, Jiangsu, Peoples R China; Childrens Hosp Nanjing Med Univ, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210008, Jiangsu, Peoples R China
ABSTRACT: Background: The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the mo-lecular and immunochemical properties of this novel allergen.Methods: The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry.
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One Size Fits All—Venomics of the Iberian Adder (Vipera seoanei, Lataste 1878) Reveals Low Levels of Venom Variation across Its Distributional Range
Toxins. 2023. Avella et al. CIBIO, BIOPOLIS Program Genom Biodivers & Land Planning, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Ctr Invest Biodiversidade & Recursos Genet, InBIO Lab Associado, CIBIO, Campus Vairao, P-4485661 Vairao, Portugal; Univ Porto, Fac Ciencias, Dept Biol, P-4099002 Porto, Portugal
ABSTRACT: European vipers (genus Vipera) are medically important snakes displaying considerable venom variation, occurring at different levels in this group. The presence of intraspecific venom variation, however, remains understudied in several Vipera species. Vipera seoanei is a venomous snake endemic to the northern Iberian Peninsula and south-western France, presenting notable phenotypic variation and inhabiting several diverse habitats across its range. We analysed the venoms of 49 adult specimens of V.
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A Cosine-Similarity-Based Deconvolution Method for Analyzing Data-Independent Acquisition Mass Spectrometry Data
APPLIED SCIENCES-BASEL. 2023. Xiang Zhang et al. Shandong Univ Technol, Sch Comp Sci & Technol, Zibo 255049, Peoples R China
ABSTRACT: Although data-independent acquisition (DIA) has the ability to identify and quantify all peptides in a sample, highly complex mixed mass spectra present difficulties for accurate peptide and protein identification. Additionally, the correspondence between the precursor and its fragments is broken, making it challenging to perform peptide identification directly using conventional DDA search engines. In this paper, we propose a cosine-similarity-based deconvolution method: CorrDIA. This is achieved by reconstructing the correspondence between precursor and fragment ions based on the consistency of extracted ion chromatograms (XICs).
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Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma
The Journal of biological chemistry. 2023. Yan, Sheng et al. Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, China
ABSTRACT: The O-linked beta-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit.
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Large-scale qualitative and quantitative assessment of dityrosine crosslinking omics in response to endogenous and exogenous hydrogen peroxide in Escherichia coli
Antioxidants. 2023. Zhou, Xiangzhe et al. School of Life Science, Beijing Institute of Technology, Beijing 100081, China
ABSTRACT:
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IgE recognition and structural analysis of disulfide bond rearrangement and chemical modifications in allergen aggregations in roasted peanuts
Journal of Agricultural and Food Chemistry. 2023. Ying Zhang et al. Nanchang Univ, Sino German Joint Res Inst, Nanchang 330047, Peoples R China; Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China
ABSTRACT: Giventhat roasting changes the structure and allergenicity ofpeanut allergens, the structural information of peanut allergens mustbe expounded to explain the alteration in their allergenicity. Thiswork focused on allergen aggregations (AAs) in roasted peanuts. IgErecognition capability was assessed via western blot analysis. Thedisulfide bond (DB) rearrangement and chemical modification in AAswere identified by combining mass spectroscopy and software tools,and structural changes induced by cross-links were displayed by moleculardynamics and PyMOL software.
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Structural surfaceomics reveals an AML-specific conformation of integrin $\beta$2 as a CAR T cellular therapy target
Nature Cancer. 2023. Kamal Manda et al. Arun P. Wiita
ABSTRACT: Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'.
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DIAPH1-MFN2 interaction regulates mitochondria-SR/ER contact and modulates ischemic/hypoxic stress
Nature Communications. 2023. Gautham Yepuri et al. Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, NYU Grossman School of Medicine, New York, New York, 10016, USA
ABSTRACT: Inter-organelle contact and communication between mitochondria and sarco/endoplasmic reticulum (SR/ER) maintain cellular homeostasis and are profoundly disturbed during tissue ischemia. We tested the hypothesis that the formin Diaphanous-1 (DIAPH1), which regulates actin dynamics, signal transduction and metabolic functions, contributes to these processes. We demonstrate that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to shorten mitochondria-SR/ER distance, thereby enhancing mitochondria-ER contact in cells including cardiomyocytes, endothelial cells and macrophages.
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A noncanonical function of SKP1 regulates the switch between autophagy and unconventional secretion
Science Advances. 2023. Li Jie et al. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA.
ABSTRACT: Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr(131), allowing its interaction with V-1 subunits of the vacuolar ATPase (V-ATPase).
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The Intrinsically Disordered N Terminus in Atg12 from Yeast Is Necessary for the Functional Structure of the Protein
International Journal of Molecular Sciences. 2023. Hana Popelka et al. Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Department of Pathology, Michigan Medicine, University of Michigan, Ann Arbor, MI 48109, USA
ABSTRACT: The Atg12 protein in yeast is an indispensable polypeptide in the highly conserved ubiquitin-like conjugation system operating in the macroautophagy/autophagy pathway. Atg12 is covalently conjugated to Atg5 through the action of Atg7 and Atg10; the Atg12-Atg5 conjugate binds Atg16 to form an E3 ligase that functions in a separate conjugation pathway involving Atg8. Atg12 is comprised of a ubiquitin-like (UBL) domain preceded at the N terminus by an intrinsically disordered protein region (IDPR), a domain that comprises a major portion of the protein but remains elusive in its conformation and function.
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The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL
FEBS Open bio. 2023. Huang Peng et al. Department of Experimental Medical Science, Lund University, Sweden
ABSTRACT: Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling.
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Architecture of the baculovirus nucleocapsid revealed by cryo-EM
Nature Communications. 2023. Jia, Xudong et al. State key laboratory of biocontrol, School of Life Sciences, Sun Yat-sen University, 510275, Guangzhou, China
ABSTRACT: Baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used as a bioinsecticide and a protein expression vector. Despite their importance, very little is known about the structure of most baculovirus proteins. Here, we show a 3.2A resolution structure of helical cylindrical body of the AcMNPV nucleocapsid, composed of VP39, as well as 4.3A resolution structures of both the head and the base of the nucleocapsid composed of over 100 protein subunits. AcMNPV VP39 demonstrates some features of the HK97-like fold and utilizes disulfide-bonds and a set of interactions at its C-termini to mediate nucleocapsid assembly and stability.
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Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome
. 2023. Xuyan Shi et al. National Institute of Biological Sciences, Beijing, Beijing, P. R. China National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, P. R. China Research Unit of Pyroptosis and Immunity, Chinese Academy of Medical Sciences and National Institute of Biological Sciences, Beijing, Beijing, P. R. China Changping Laboratory, Beijing, P. R. China Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, P. R. China New Cornerstone Science Laboratory, Shenzhen, P. R. China
ABSTRACT:
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Structure of the transcribing RNA polymerase II--Elongin complex
Nature Structural & Molecular Biology. 2023. Chen, Ying et al. Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen,
ABSTRACT: Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-ELOC subunit heterodimer. ELOA contains a 'latch' that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center.
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The LIKE SEX FOUR 1--malate dehydrogenase complex functions as a scaffold to recruit $\beta$-amylase to promote starch degradation
The Plant Cell. 2023. Jian Liu et al. Huazhong Agr Univ, Natl Ctr Plant Gene Res, Wuhan 430070, Peoples R China; Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan 430070, Peoples R China
ABSTRACT: In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including beta-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit beta-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex.
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Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane
Nature Structural & Molecular Biology. 2023. Park, Si Hoon et al. Department of Life Sciences, Korea University, Seoul, South Korea
ABSTRACT: Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity.
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Peroxiredoxin-1 is an H2O2 safe-guard antioxidant and signalling enzyme in M1 macrophages
. 2023. Ezeri{\c{n}}a, Daria et al. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels B-1050, Belgium
ABSTRACT:
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ECL 3.0: a sensitive peptide identification tool for cross-linking mass spectrometry data analysis
BMC bioinformatics. 2023. Zhou, Chen et al. Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Pleinlaan 2, Brussels B-1050, Belgium
ABSTRACT: BACKGROUND: Cross-linking mass spectrometry (XL-MS) is a powerful technique for detecting protein-protein interactions (PPIs) and modeling protein structures in a high-throughput manner. In XL-MS experiments, proteins are cross-linked by a chemical reagent (namely cross-linker), fragmented, and then fed into a tandem mass spectrum (MS/MS). Cross-linkers are either cleavable or non-cleavable, and each type requires distinct data analysis tools. However, both types of cross-linkers suffer from imbalanced fragmentation efficiency, resulting in a large number of unidentifiable spectra that hinder the discovery of PPIs and protein conformations.
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4D-diaXLMS: Proteome-wide Four-Dimensional Data-Independent Acquisition Workflow for Cross-Linking Mass Spectrometry
Analytical Chemistry. 2023. Yanhong Hao et al. Suming Chen
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful tool for examining protein structures and interactions. Nevertheless, analysis of low-abundance cross-linked peptides is often limited in the data-dependent acquisition (DDA) mode due to its semistochastic nature. To address this issue, we introduced a workflow called 4D-diaXLMS, representing the first-ever application of four-dimensional data-independent acquisition for proteome-wide cross-linking analysis. Cross-linking studies of the HeLa cell proteome were evaluated using the classical cross-linker disuccinimidyl suberate as an example.
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CaMKII autophosphorylation can occur between holoenzymes without subunit exchange
Elife. 2023. Lu{\v{c}}i{\'c}, Iva et al. Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin
ABSTRACT: The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange.
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Structural basis of nucleosome deacetylation and DNA linker tightening by Rpd3S histone deacetylase complex
Cell Research. 2023. Dong, Shuqi et al. CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, GIBH-HKU Guangdong–Hong Kong Stem Cell and Regenerative Medicine Research Centre, GIBH-CUHK Joint Research Laboratory on Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, China
ABSTRACT: In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP.
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Structural insights into human co-transcriptional capping
Molecular Cell. 2023. Gaurika Garg et al. Max Planck Institute for Multidisciplinary Sciences, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany
ABSTRACT: Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end.
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Proteomic Characterization of Native and Rearranged Disulfide Bonds in Proteins from Thermally Treated and Commercial Milk Samples
Journal of Agricultural and Food Chemistry. 2023. Valentina Ciaravolo et al. CNR, Prote Metabol & Mass Spectrometry Lab, ISPAAM, I-80055 Portici, Italy
ABSTRACT: To investigate thiol-disulfide interchange reactionsin heatedmilk yielding non-native intramolecular rearranged and intermolecularcross-linked proteins, a proteomic study based on nanoLC-ESI-Q-Orbitrap-MS/MSand dedicated bioinformatics was accomplished. Raw milk samples heatedfor different times and various commercial dairy products were analyzed.Qualitative experiments on tryptic digests of resolved protein mixturesassigned the corresponding disulfide-linked peptides. Results confirmedthe limited data available on few milk proteins, generated the widestinventory of components (63 in number) involved in thiol-disulfideexchange processes, and provided novel structural information on S-S-bridgedmolecules.
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Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells
Analytical Chemistry. 2023. Xinwei Li et al. Chinese Acad Sci, Dalian Inst Chem Phys, Collaborat Innovat Ctr Chem Energy Mat iChEM, State Key Lab Catalysis, Dalian 116023, Peoples R China; Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: Photosynthesis, as the core of solar energy biotransformation,is driven by photosynthetic membrane protein complexes in plants andalgae. Current methods for intracellular photosynthetic membrane proteincomplex analysis mostly require the separation of specific chloroplastsor the change of the intracellular environment, which causes the missingof real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membraneprotein complexes in the chloroplasts of living Chlamydomonasreinhardtii (C.
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Peroxiredoxin-1 is an H2o2 Safe-Guard Antioxidant and Signalling Enzyme in Macrophages Independent of Their Polarization State
Available at SSRN 4445887. 2023. Daria Ezeriņa et al. VIB-VUB Center for Structural Biology, Vlaams Instituut Voor Biotechnologie, Brussels B-1050, Belgium
ABSTRACT:
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Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae
Probiotics and Antimicrobial Proteins. 2023. Michelle Rossouw et al. Stellenbosch Univ, Dept Microbiol, Private Bag X1, ZA-7602 Matieland, South Africa
ABSTRACT: Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX).
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In vivo cross-linking-based affinity purification and mass spectrometry for targeting intracellular protein-protein interactions
Analytica Chimica Acta. 2023. Zhong, Bowen et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: Comprehensive interactome analysis of targeted proteins is important to understand how proteins work together in regulating functions. Commonly, affinity purification followed by mass spectrometry (AP-MS) has been recognized as the most often used technique for studying protein-protein interactions (PPIs). However, some proteins with weak interactions, which are responsible for key roles in regulation, are easily broken during cell lysis and purification through an AP approach. Herein, we have developed an approach termed in vivo cross-linking-based affinity purification and mass spectrometry (ICAP-MS).
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Total Chemical Synthesis of Glycosylated TREM2 Ectodomain
ACS Chemical Neuroscience. 2023. Gayani Wijegunawardena et al. Univ Calif San Francisco, Weill Inst Neurosci, Dept Neurol, San Francisco, CA 94158 USA; Univ Calif San Francisco, Inst Neurodegenerat Dis, San Francisco, CA 94158 USA; Wichita State Univ, Dept Chem & Biochem, Wichita, KS 67260 USA
ABSTRACT: Mutations in a microglia-associated gene TREM2increase the risk of Alzheimer's disease. Currently, structuraland functional studies of TREM2 mainly rely on recombinant TREM2 proteinsexpressed from mammalian cells. However, using this method, it isdifficult to achieve site-specific labeling. Here, we present thetotal chemical synthesis of the 116 amino acid TREM2 ectodomain. Rigorousstructural analysis ensured correct structural fold after refolding.Treating microglial cells with refolded synthetic TREM2 enhanced microglialphagocytosis, proliferation, and survival.
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Near-atomic architecture of Singapore grouper iridovirus and implications for giant virus assembly
Nature Communications. 2023. Zhennan Zhao et al. Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China; Southern Univ Sci & Technol, Cryo EM Ctr, Dept Biol, Shenzhen 518055, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, Clin Res Inst, State Key Lab Complex Severe & Rare Dis, Beijing 100730, Peoples R China; South China Agr Univ, Coll Marine Sci, Guangdong Lab Lingnan Modern Agr, Guangzhou 510642, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Inst Microbiol, CAS Key Lab Pathogen Microbiol & Immunol, Beijing 100101, Peoples R China
ABSTRACT: High morbidity and mortality in aquatic have been caused by iridovirids worldwide. Here the authors present a near-atomic SGIV capsid structure. Functional assays further reveal the relationships between identified capsid proteins and viral assembly.Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide.
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Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in Saccharomyces cerevisiae
Nucleic Acids Research. 2023. Ellison, Mitchell A. et al. Univ Pittsburgh, Dept Biol Sci, Pittsburgh, PA 15260 USA
ABSTRACT: The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes.
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ERR$\gamma$-DBD undergoes dimerization and conformational rearrangement upon binding to the downstream site of the DR1 element
Biochemical and Biophysical Research Communications. 2023. Xuhui Zhen  et al. Chinese Acad Sci, Guangzhou Inst Hlth, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China; Chinese Acad Sci, Guangzhou Inst Biomed, Guangdong Prov Key Lab Biocomp, Guangzhou 510530, Peoples R China
ABSTRACT: The estrogen-related receptor (ERR) family members are reported to bind DNA elements as either monomer or dimer. However, to date, only one solution NMR structure of ERRI3 in complex with a half -site DNA element has been reported. To better understand the DNA regulation mechanism, we deter-mined the crystal structure of ERRy-DBD bound to a natural DR1 element in Pla2g12b promoter to 2.2 angstrom resolution. Combined with biochemical assays, we show that ERRy acts as a dimer and the C-terminal extension region undergoes conformational rearrangement when binding to the downstream DR1 element.
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Regulation of 3′ splice site selection after step 1 of splicing by spliceosomal C* proteins
Science Advances. 2023. Dybkov, Olexandr et al. Max Planck Inst Multidisciplinary Sci, Cellular Biochem, Fassberg 11, D-37077 Gottingen, Germany; Free Univ Berlin, Inst Chem & Biochem, RNA Biochem, Takustr 6, D-14195 Berlin, Germany
ABSTRACT: Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3 ' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spli-ceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3 ' ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage.
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Structural basis of bacterial effector protein azurin targeting tumor suppressor p53 and inhibiting its ubiquitination
Communications Biology. 2023. Hu, Jianjian et al. Hubei Univ, Coll Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Wuhan 430074, Peoples R China
ABSTRACT: Structural and mutagenic analyses reveal the mechanistic basis of azurin-mediated p53 stabilization and tumor suppression, and several affinity-enhancing azurin mutants are designed.Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level.
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IgE Recognition and Structural Analysis of Disulfide Bond Rearrangement and Chemical Modifications in Allergen Aggregations in Roasted Peanuts
Journal of Agricultural and Food Chemistry. 2023. Ying Zhang et al. Nanchang Univ, Sino German Joint Res Inst, Nanchang 330047, Peoples R China; Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China
ABSTRACT: Giventhat roasting changes the structure and allergenicity ofpeanut allergens, the structural information of peanut allergens mustbe expounded to explain the alteration in their allergenicity. Thiswork focused on allergen aggregations (AAs) in roasted peanuts. IgErecognition capability was assessed via western blot analysis. Thedisulfide bond (DB) rearrangement and chemical modification in AAswere identified by combining mass spectroscopy and software tools,and structural changes induced by cross-links were displayed by moleculardynamics and PyMOL software.
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Structural basis of amine odorant perception by a mammal olfactory receptor
Nature. 2023. Lulu Guo et al. Shanghai Jiao Tong Univ, Ctr Brain Sci, Shanghai Childrens Med Ctr, Sch Med, Shanghai, Peoples R China; Shandong Univ, Adv Med Res Inst, Cheeloo Coll Med, Meili Lake Translat Res Pk, Jinan, Peoples R China; Shandong Univ, Dept Gen Surg, Qilu Hosp, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Inst, Sch Med, Shanghai, Peoples R China; Shandong Univ, Dept Biochem & Mol Biol, Sch Med, Jinan, Peoples R China; Shanghai Jiao Tong Univ, Songjiang Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Res Ctr Brain Sci & Brain Inspired Intel, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Shanghai Key Lab Childrens Environm Hlth, Dept Anat & Physiol, Minist Educ,Sch Med,Xinhua Hosp, Shanghai, Peoples R China; Peking Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Key Lab Mol Cardiovasc Sci,Minist Educ, Beijing, Peoples R China
ABSTRACT: Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with beta-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine.
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Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM
Communications Biology. 2023. Caixuan Liu et al. Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol,Natl Ctr Prot Sci Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC.
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Large-Scale Qualitative and Quantitative Assessment of Dityrosine Crosslinking Omics in Response to Endogenous and Exogenous Hydrogen Peroxide in Escherichia coli
Antioxidants. 2023. Xiangzhe Zhou et al. Beijing Inst Technol, Sch Life Sci, Beijing 100081, Peoples R China; Beijing Inst Technol, Inst Engn Med, Sch Med Technol, Beijing 100081, Peoples R China
ABSTRACT: Excessive hydrogen peroxide causes oxidative stress in cells. The oxidation of two tyrosine residues in proteins can generate o,o '-dityrosine, a putative biomarker for protein oxidation, which plays critical roles in a variety of organisms. Thus far, few studies have investigated dityrosine crosslinking under endogenous or exogenous oxidative conditions at the proteome level, and its physiological function remains largely unknown. In this study, to investigate qualitative and quantitative dityrosine crosslinking, two mutant Escherichia coli strains and one mutant strain supplemented with H2O2 were used as models for endogenous and exogenous oxidative stress, respectively.
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Diverse modes of H3K36me3-guided nucleosomal deacetylation by Rpd3S
Nature. 2023. Guan, Haipeng et al. State Key Laboratory of Molecular Oncology, MOE Key Laboratory of Protein Sciences, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, School of Medicine, Tsinghua University, Beijing, China
ABSTRACT: Context-dependent dynamic histone modifications constitute a key epigenetic mechanism in gene regulation1-4. The Rpd3 small (Rpd3S) complex recognizes histone H3 trimethylation on lysine 36 (H3K36me3) and deacetylates histones H3 and H4 at multiple sites across transcribed regions5-7. Here we solved the cryo-electron microscopy structures of Saccharomyces cerevisiae Rpd3S in its free and H3K36me3 nucleosome-bound states. We demonstrated a unique architecture of Rpd3S, in which two copies of Eaf3-Rco1 heterodimers are asymmetrically assembled with Rpd3 and Sin3 to form a catalytic core complex.
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Hydrogen sulfide functions as a micro-modulator bound at the copper active site of Cu/Zn-SOD to regulate the catalytic activity of the enzyme
Cell Reports. 2023. Dongdong Wu et al. Univ Shanghai Sci & Technol, Sch Hlth Sci & Engn, Shanghai 200093, Peoples R China; Chinese Acad Sci, Shenzhen Inst Synthet Biol, Shenzhen Inst Adv Technol, CAS Key Lab Quantitat Engn Biol, Shenzhen 518055, Peoples R China; Fudan Univ, Sch Basic Med Sci, Dept Physiol & Pathophysiol, Shanghai Key Lab Bioact Small Mol,Shanghai Med Col, Shanghai 200032, Peoples R China; Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Chem Biol, Shanghai 200032, Peoples R China
ABSTRACT: The present study examines whether there is a mechanism beyond the current concept of post-translational modifications to regulate the function of a protein. A small gas molecule, hydrogen sulfide (H2S), was found to bind at active-site copper of Cu/Zn-SOD using a series of methods including radiolabeled binding assay, X-ray absorption near-edge structure (XANES), and crystallography. Such an H2S binding enhanced the elec-trostatic forces to guide the negatively charged substrate superoxide radicals to the catalytic copper ion, changed the geometry and energy of the frontier molecular orbitals of the active site, and subsequently facil-itated the transfer of an electron from the superoxide radical to the catalytic copper ion and the breakage of the copper-His61 bridge.
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Structural basis of mRNA binding by the human FERRY Rab5 effector complex
Molecular Cell. 2023. Jan Schuhmacher et al. Max Planck Inst Mol Physiol, Dept Struct Biochem, D-44227 Dortmund, Germany; Max Planck Inst Mol Cell Biol & Genet, D-01307 Dresden, Germany
ABSTRACT: The pentameric FERRY Rab5 effector complex is a molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of human FERRY. It reveals a unique clamp-like architecture that bears no resemblance to any known structure of Rab effectors. A com-bination of functional and mutational studies reveals that while the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly.
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Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells
ACS Chemical Biology. 2023. Shengrong Li et al. Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, MOE Key Lab Tumor Mol Biol, Guangzhou 510632, Peoples R China; Jinan Univ, Sch Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat & I, Guangzhou 510632, Peoples R China
ABSTRACT: Chemicalcross-linking of proteins coupled with massspectrometryanalysis (CXMS) is a powerful method for the study of protein structureand protein-protein interactions (PPIs). However, the chemicalprobes used in the CXMS are limited to bidentate reactive warheads,and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS)and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonylynamide, was developed as a new zero-length cross-linker that canconnect high-abundance carboxyl residues (D/E) with lysine (K) toform amide bonds in the absence of any catalyst.
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Structure of a SIN3--HDAC complex from budding yeast
Nature Structural & Molecular Biology. 2023. Zhouyan Guo et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Peoples R China
ABSTRACT: SIN3-HDAC (histone deacetylases) complexes have important roles in facilitating local histone deacetylation to regulate chromatin accessibility and gene expression. Here, we present the cryo-EM structure of the budding yeast SIN3-HDAC complex Rpd3L at an average resolution of 2.6 angstrom. The structure reveals that two distinct arms (ARM1 and ARM2) hang on a T-shaped scaffold formed by two coiled-coil domains. In each arm, Sin3 interacts with different subunits to create a different environment for the histone deacetylase Rpd3.
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Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly
Communications Biology. 2023. Hesselbarth, Julia et al. Martin Luther Univ Halle Wittenberg, Inst Biochem & Biotechnol, Interdisciplinary Res Ctr HALOmem, Charles Tanford Prot Ctr, Halle, Germany; Johannes Gutenberg Univ Mainz, Dept Chem Biochem, Bioctr 2, Mainz, Germany
ABSTRACT: The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1.
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Structural insights into DNA N6-adenine methylation by the MTA1 complex
Cell Discovery. 2023. Yan, Junjun et al. Huazhong Agr Univ, Natl Key Lab Crop Genet Improvement, Wuhan, Hubei, Peoples R China; Huazhong Agr Univ, Natl Ctr Plant Gene Res, Hubei Hongshan Lab, Wuhan, Hubei, Peoples R China; Hubei Univ, Hubei Collaborat Innovat Ctr Green Transformat Bio, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn,Hubei Key, Wuhan, Hubei, Peoples R China
ABSTRACT: N-6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 angstrom) and S-adenosyl homocysteine (SAH)-bound (2.8 angstrom) states.
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Fine structure and assembly pattern of a minimal myophage Pam3
Proceedings of the National Academy of Sciences of the United States of America. 2023. Feng Yang et al. School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
ABSTRACT: The myophage possesses a contractile tail that penetrates its host cell envelope. Except for investigations on the bacteriophage T4 with a rather complicated structure, the assembly pattern and tail contraction mechanism of myophage remain largely unknown. Here, we present the fine structure of a freshwater Myoviridae cyanophage Pam3, which has an icosahedral capsid of ~680 A in diameter, connected via a three-section neck to an 840-A-long contractile tail, ending with a three-module baseplate composed of only six protein components.
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Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline
Journal of Proteome Research. 2023. Hoopmann, Michael R. et al. Inst Syst Biol, Seattle, WA 98109 USA
ABSTRACT: Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here.
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ERR$\gamma$-DBD undergoes dimerization and conformational rearrangement upon binding to a DR1 element in Pla2g12b promoter
. 2023. Zhen, Xuhui et al. Guangdong Provincial Key Laboratory of Biocomputing, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
ABSTRACT:
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Optimized approach for active peptides identification in Cerebrolysin by nanoLC-MS
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES. 2023. Bingkun Yang et al. Hebei Med Univ, Inst Chinese Integrat Med, Sch Chinese Integrat Med, Dept Pharmacol Chinese Mat Med, 361,Zhongshan East Rd, Shijiazhuang 050017, Hebei, Peoples R China; Hebei Med Univ, Sch Pharm, Shijiazhuang 050017, Peoples R China
ABSTRACT: Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neuro-trophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent).
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Comprehensive evaluation of peptide de novo sequencing tools for monoclonal antibody assembly
Briefings in Bioinformatics. 2023. Beslic, Denis et al. BAM Fed Inst Mat Res & Testing, Richard Willstatter Str 11, D-12489 Berlin, Germany; Univ Potsdam, Digital Engn Fac, Hasso Plattner Inst, Prof Dr Helmert Str 2-3, D-14482 Potsdam, Germany; BAM Fed Inst Mat Res & Testing, Unter Eichen 87, D-12205 Berlin, Germany; Robert Koch Inst, ZKI PH 3,Nordufer 20, D-13353 Berlin, Germany
ABSTRACT: Monoclonal antibodies are biotechnologically produced proteins with various applications in research, therapeutics and diagnostics. Their ability to recognize and bind to specific molecule structures makes them essential research tools and therapeutic agents. Sequence information of antibodies is helpful for understanding antibody-antigen interactions and ensuring their affinity and specificity. De novo protein sequencing based on mass spectrometry is a valuable method to obtain the amino acid sequence of peptides and proteins without a priori knowledge.
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GlycoTCFM: Glycoproteomics Based on Two Complementary Fragmentation Methods Reveals Distinctive O-Glycosylation in Human Sperm and Seminal Plasma
Journal of Proteome Research. 2023. Mengqi Luo et al. Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041,
ABSTRACT: Human semen, consisting of spermatozoa (sperm) and seminal plasma, represents a special clinical sample type in human body fluid. Protein glycosylation in sperm and seminal plasma plays key roles in spermatogenesis, maturation, capacitation, sperm-egg recognition, motility of sperm, and fertilization. In this study, we profiled the most comprehensive O-glycoproteome map of human sperm and seminal plasma using our recently presented Glycoproteomics based on Two Complementary Fragmentation Methods (GlycoTCFM).
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An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments
Proteomics. 2023. Chu-Wei Kuo et al. Acad Sinica, Inst Biol Chem, 128 Acad Rd Sec 2, Taipei 115, Taiwan
ABSTRACT: Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin.
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Use: pGlyco



Expression of a Siglec-Fc Protein and Its Characterization
BIOLOGY-BASEL. 2023. Kaijun Chi et al. Jiangnan Univ, Sch Biotechnol, Key Lab Carbohydrate Chem & Biotechnol, Minist Educ, Wuxi 214122, Peoples R China; Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
ABSTRACT: Simple Summary The Siglec-Fc protein, a fusion protein combining Siglec with the Fc part of a human antibody, is a promising sialic acid-Siglec axis-targeted agent for cancer treatment and is widely used for Siglec ligands discovery. The recombinant Siglec-Fc fusion protein has been expressed in different cell systems. However, its characteristics have not been investigated in detail. In this study, HEK293 and CHO cell lines were used to express the Siglec9-Fc protein, and their adaptability for production was compared.
[more...]
Use: pGlyco



Glycopeptide database search and de novo sequencing with PEAKS GlycanFinder enable highly sensitive glycoproteomics
Nature Communications. 2023. Sun, Weiping et al. Bioinformatics Solutions Inc., Waterloo, Ontario, Canada
ABSTRACT: Here we present GlycanFinder, a database search and de novo sequencing tool for the analysis of intact glycopeptides from mass spectrometry data. GlycanFinder integrates peptide-based and glycan-based search strategies to address the challenge of complex fragmentation of glycopeptides. A deep learning model is designed to capture glycan tree structures and their fragment ions for de novo sequencing of glycans that do not exist in the database. We performed extensive analyses to validate the false discovery rates (FDRs) at both peptide and glycan levels and to evaluate GlycanFinder based on comprehensive benchmarks from previous community-based studies.
[more...]
Use: pGlyco; pDeep



Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC--HRMS
Scientific Reports. 2023. Yoondam Seo et al. Doping Control Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil, Seongbuk-gu, Seoul, 02792, Republic of Korea
ABSTRACT: Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood's capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure.
[more...]
Use: pGlyco



High-coverage four-Dimensional data-independent acquisition proteomics and phosphoproteomics enabled by deep learning-driven multidimensional predictions
Analytical Chemistry. 2023. Moran Chen et al. Wuhan Univ, Inst Adv Studies, Wuhan 430072, Hubei, Peoples R China
ABSTRACT: Four-dimensional (4D) data-independentacquisition (DIA)-basedproteomics is a promising technology. However, its full performanceis restricted by the time-consuming building and limited coverageof a project-specific experimental library. Herein, we developed aversatile multifunctional deep learning model Deep4D based on self-attentionthat could predict the collisional cross section, retention time,fragment ion intensity, and charge state with high accuracies forboth the unmodified and phosphorylated peptides and thus establishedthe complete workflows for high-coverage 4D DIA proteomics and phosphoproteomicsbased on multidimensional predictions.
[more...]
Use: pDeep



Test-Time Training for Deep MS/MS Spectrum Prediction Improves Peptide Identification
Journal of Proteome Research. 2023. Jianbai Ye et al. MoE Key Laboratory of Brain-inspired Intelligent Perception and Cognition, University of Science and Technology of China, Hefei, Anhui 230026, China
ABSTRACT:
Use: pDeep



AIomics: exploring more of the proteome using mass spectral libraries extended by artificial intelligence
Journal of Proteome Research. 2023. Lewis Y. Geer et al. Natl Inst Stand & Technol, Mass Spectrometry Data Ctr, Biomol Measurement Div, Gaithersburg, MD 20899 USA
ABSTRACT: The unbounded permutations of biological molecules, includingproteinsand their constituent peptides, present a dilemma in identifying thecomponents of complex biosamples. Sequence search algorithms usedto identify peptide spectra can be expanded to cover larger classesof molecules, including more modifications, isoforms, and atypicalcleavage, but at the cost of false positives or false negatives dueto the simplified spectra they compute from sequence records. Spectrallibrary searching can help solve this issue by precisely matchingexperimental spectra to library spectra with excellent sensitivityand specificity.
[more...]
Use: pDeep



Machine learning-based peptide-spectrum match rescoring opens up the immunopeptidome
. 2023. Charlotte Adams et al. Laboratory of Protein Science, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Antwerp,
ABSTRACT:
Use: pDeep



High-Coverage Four-Dimensional Data-Independent Acquisition Proteomics and Phosphoproteomics Enabled by Deep Learning-Driven Multidimensional Predictions
Analytical Chemistry. 2023. Moran Chen et al. Wuhan Univ, Inst Adv Studies, Wuhan 430072, Hubei, Peoples R China
ABSTRACT: Four-dimensional (4D) data-independentacquisition (DIA)-basedproteomics is a promising technology. However, its full performanceis restricted by the time-consuming building and limited coverageof a project-specific experimental library. Herein, we developed aversatile multifunctional deep learning model Deep4D based on self-attentionthat could predict the collisional cross section, retention time,fragment ion intensity, and charge state with high accuracies forboth the unmodified and phosphorylated peptides and thus establishedthe complete workflows for high-coverage 4D DIA proteomics and phosphoproteomicsbased on multidimensional predictions.
[more...]
Use: pDeep



AIomics: Exploring More of the Proteome Using Mass Spectral Libraries Extended by Artificial Intelligence
Journal of Proteome Research. 2023. Lewis Y. Geer et al. Natl Inst Stand & Technol, Mass Spectrometry Data Ctr, Biomol Measurement Div, Gaithersburg, MD 20899 USA
ABSTRACT: The unbounded permutations of biological molecules, includingproteinsand their constituent peptides, present a dilemma in identifying thecomponents of complex biosamples. Sequence search algorithms usedto identify peptide spectra can be expanded to cover larger classesof molecules, including more modifications, isoforms, and atypicalcleavage, but at the cost of false positives or false negatives dueto the simplified spectra they compute from sequence records. Spectrallibrary searching can help solve this issue by precisely matchingexperimental spectra to library spectra with excellent sensitivityand specificity.
[more...]
Use: pDeep




2022




Binding stoichiometry and structural model of the HIV-1 Rev/Importin $\beta$ complex
Life Science Alliance. 2022. Spittler, Didier et al. Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, USA
ABSTRACT: HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin beta (Impbeta), but how Rev associates with Impbeta is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Impbeta/Rev complex. We show that Impbeta binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Impbeta cargos.
[more...]
Use: pFind; pDeep; pLink



Nanobodies and chemical cross-links advance the structural and functional analysis of PI3K$\alpha$
Proceedings of the National Academy of Sciences of the United States of America. 2022. Hart, Jonathan R et al. Department of Molecular Medicine, Scripps Research Institute, La Jolla, CA 92037
ABSTRACT: Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kalpha. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kalpha catalytic core. Binding of individual nanobodies to PI3Kalpha induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function.
[more...]
Use: pFind; pDeep; pLink



INAUGURAL ARTICLE by a Recently Elected Academy Member: Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome
Proceedings of the National Academy of Sciences of the United States of America. 2022. Rahman, Sanim et al. Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
ABSTRACT:
Use: pFind; pDeep; pLink



Integrative analysis reveals structural basis for transcription activation of Nurr1 and Nurr1-RXR$\alpha$ heterodimer
Proceedings of the National Academy of Sciences of the United States of America. 2022. Zhao, Mohan et al. State Key Laboratory of Respiratory Disease, Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
ABSTRACT: Orphan nuclear receptor Nurr1 plays important roles in the progression of various diseases, including Parkinson's disease, neuroinflammation, Alzheimer's disease, and multiple sclerosis. It can recognize DNA as a monomer or heterodimer with retinoid X receptor alpha (RXRalpha). But the molecular mechanism of its transcriptional activity regulation is still largely unknown. Here we obtained a crystal structure of monomer Nurr1 (DNA- and ligand-binding domains, DBD and LBD) bound to NGFI-B response element.
[more...]
Use: pFind; pDeep; pLink



Immune checkpoint therapy-elicited sialylation of IgG antibodies impairs antitumorigenic type I interferon responses in hepatocellular carcinoma
Immunity. 2022. Wu, Rui-Qi et al. Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Oncology in South China
ABSTRACT: The reinvigoration of anti-tumor Tcells in response to immune checkpoint blockade (ICB) therapy is well established. Whether and how ICB therapy manipulates antibody-mediated immune response in cancer environments, however, remains elusive. Using tandem mass spectrometric analysis of modification of immunoglobulin G (IgG) from hepatoma tissues, we identified a role of ICB therapy in catalyzing IgG sialylation in the Fc region. Effector Tcells triggered sialylation of IgG via an interferon (IFN)-gamma-ST6Gal-I-dependent pathway.
[more...]
Use: pFind; pDeep; pGlyco



Mechanistic insights into the functioning of a two-subunit GMP synthetase, an allosterically regulated, ammonia channeling enzyme
BIOCHEMISTRY. 2022. Shivakumaraswamy, S et al. Jawaharlal Nehru Ctr Adv Sci Res, Mol Biol & Genet Unit, Bengaluru 560064, India
ABSTRACT: Guanosine 5 & PRIME;-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 & PRIME;-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP.
[more...]
Use: pFind; pLink



Efficient detection of the alternative spliced Human proteome using translatome sequencing
Frontiers in Molecular Biosciences. 2022. Wu, Chun et al. Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China
ABSTRACT: Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms.
[more...]
Use: pFind



MetaLab-MAG: A metaproteomic data analysis platform for genome-level characterization of microbiomes from the metagenome-assembled genomes database
Journal of Proteome Research. 2022. Cheng, Kai et al. Univ Ottawa, Fac Med, Sch Pharmaceut Sci, Ottawa, ON K1H 8M5, Canada
ABSTRACT: The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases.
[more...]
Use: pFind



Discovery of 194 Unreported Conopeptides and Identification of a New Protein Disulfide Isomerase in Conus caracteristicus Using Integrated Transcriptomic and Proteomic Analysis
Frontiers in Marine Science. 2022. Han Zhang1 et al. department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
ABSTRACT: Current ConoServer database accumulates 8,134 conopeptides from 122 species of cone snail, which are pharmaceutically attractive marine resource. However, many more conopeptides remain to be discovered, and the enzymes involved in their synthesis and processing are unclear. In this report, firstly we screened and analyzed the differentially expressed genes (DEGs) between venom duct (VD) and venom bulb (VB) of C. caracteristicus, and obtained 3,289 transcripts using a comprehensive assembly strategy.
[more...]
Use: pFind



A hybrid spectral library and protein sequence database search strategy for bottom-up and top-down proteomic data analysis
Journal of Proteome Research. 2022. Dai, YL et al. Univ Wisconsin, Dept Chem, Madison, WI 53706 USA
ABSTRACT: Tandem mass spectrometry (MS/MS) is widely employed for the analysis of complex proteomic samples. While protein sequence database searching and spectral library searching are both well-established peptide identification methods, each has shortcomings. Protein sequence databases lack fragment peak intensity information, which can result in poor discrimination between correct and incorrect spectrum assignments. Spectral libraries usually contain fewer peptides than protein sequence databases, which limits the number of peptides that can be identified.
[more...]
Use: pFind; pDeep



A protocol of using PTMiner for quality control and localization of protein modifications identified by open or closed search of tandem mass spectra
Biophysics Reports. 2022. Cheng, Zhiyuan et al. Academy of Mathematics and Systems Science, Chinese Academy of Sciences, Beijing 100190, China
ABSTRACT: In recent years, an open search of tandem mass spectra has greatly promoted the detection of post-translational modifications (PTMs) in shotgun proteomics. However, post-processing of the results from open searches remains an unsatisfactorily resolved problem, which hinders the open search mode from wide practical use. PTMiner is a software tool based on dedicated statistical algorithms for reliable filtering, localization and annotation of the modifications (mass shifts) detected by open search.
[more...]
Use: pFind; pDeep



Identification and mechanism of G protein-biased ligands for chemokine receptor CCR1
Nature chemical biology. 2022. Shao, ZH et al. Zhejiang Univ, MOE Frontier Sci Ctr Brain Res & Brain Machine In, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Pharmacol, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Dept Pathol, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 2, Dept Resp & Crit Care Med, Key Lab Resp Dis Zhejiang Prov,Sch Med, Hangzhou, Peoples R China; Zhejiang Prov Key Lab Immun & Inflammatory Dis, Hangzhou, Peoples R China; Zhejiang Univ, Key Lab Resp Dis Zhejiang Prov, Dept Resp & Crit Care Med, Affiliated Hosp 2,Sch Med, Hangzhou, Peoples R China; State Key Lab Resp Dis, Guangzhou, Peoples R China; Zhejiang Univ, Liangzhu Lab, Med Ctr, Hangzhou, Peoples R China; Zhejiang Univ, Int Inst Med, Sch Med, Affiliated Hosp 4, Yiwu, Peoples R China; Zhejiang Univ, Dept Biophys, Sir Run Run Shaw Hosp, Sch Med, Hangzhou, Peoples R China
ABSTRACT: Biased signaling of G protein-coupled receptors describes an ability of different ligands that preferentially activate an alternative downstream signaling pathway. In this work, we identified and characterized different N-terminal truncations of endogenous chemokine CCL15 as balanced or biased agonists targeting CCR1, and presented three cryogenic-electron microscopy structures of the CCR1-G(i) complex in the ligand-free form or bound to different CCL15 truncations with a resolution of 2.6-2.9 angstrom, illustrating the structural basis of natural biased signaling that initiates an inflammation response.
[more...]
Use: pFind



MStoCIRC: A powerful tool for downstream analysis of MS/MS data to predict translatable circRNAs
Frontiers in Molecular Biosciences. 2022. Cao, Zhou et al. Shaanxi Normal Univ, Coll Life Sci, Key Lab Minist Educ Med Plant Resource & Nat Pharm, Natl Engn Lab Resource Dev Endangered Crude Drugs, Xian, Peoples R China
ABSTRACT: CircRNAs are formed by a non-canonical splicing method and appear circular in nature. CircRNAs are widely distributed in organisms and have the features of time- and tissue-specific expressions. CircRNAs have attracted increasing interest from scientists because of their non-negligible effects on the growth and development of organisms. The translation capability of circRNAs is a novel and valuable direction in the functional research of circRNAs. To explore the translation potential of circRNAs, some progress has been made in both experimental identification and computational prediction.
[more...]
Use: pFind



A Hybrid Spectral Library and Protein Sequence Database Search Strategy for Bottom-Up and Top-Down Proteomic Data Analysis
Journal of Proteome Research. 2022. Dai, YL et al. Univ Wisconsin, Dept Chem, Madison, WI 53706 USA
ABSTRACT: Tandem mass spectrometry (MS/MS) is widely employed for the analysis of complex proteomic samples. While protein sequence database searching and spectral library searching are both well-established peptide identification methods, each has shortcomings. Protein sequence databases lack fragment peak intensity information, which can result in poor discrimination between correct and incorrect spectrum assignments. Spectral libraries usually contain fewer peptides than protein sequence databases, which limits the number of peptides that can be identified.
[more...]
Use: pFind; pDeep



High-throughput proteomic sample preparation using pressure cycling technology
Nature protocols. 2022. Cai, X et al. Westlake Univ, Sch Life Sci, Westlake Lab Life Sci & Biomed, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Peoples R China
ABSTRACT: High-throughput lysis and proteolytic digestion of biopsy-level tissue specimens is a major bottleneck for clinical proteomics. Here we describe a detailed protocol of pressure cycling technology (PCT)-assisted sample preparation for proteomic analysis of biopsy tissues. A piece of fresh frozen or formalin-fixed paraffin-embedded tissue weighing similar to 0.1-2 mg is placed in a 150 mu L pressure-resistant tube called a PCT-MicroTube with proper lysis buffer. After closing with a PCT-MicroPestle, a batch of 16 PCT-MicroTubes are placed in a Barocycler, which imposes oscillating pressure to the samples from one atmosphere to up to similar to 3,000 times atmospheric pressure.
[more...]
Use: pFind



Probing strigolactone perception mechanisms with rationally designed small-molecule agonists stimulating germination of root parasitic weeds
Nature communications. 2022. Wang, DW et al. Hunan Univ, State Key Lab Chemo Biosensing & Chemometr, Hunan Prov Key Lab Plant Funct Genom & Dev Regula, Coll Biol, Changsha 410082, Peoples R China; Nankai Univ, Collaborat Innovat Ctr Chem Sci & Engn, Natl Pesticide Engn Res Ctr, Coll Chem,Dept Chem Biol, Tianjin 300071, Peoples R China; Univ Amsterdam, Swammerdam Inst Life Sci SILS, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands; Nankai Univ, State Key Lab Elementoorgan Chem, Collaborat Innovat Ctr Chem Sci & Engn, Coll Chem,Natl Pesticide Engn Res Ctr, Tianjin 300071, Peoples R China
ABSTRACT: The development of potent strigolactone (SL) agonists as suicidal germination inducers could be a useful strategy for controlling root parasitic weeds, but uncertainty about the SL perception mechanism impedes real progress. Here we describe small-molecule agonists that efficiently stimulate Phelipanchce aegyptiaca, and Striga hermonthica, germination in concentrations as low as 10(-8) to 10(-17) M. We show that full efficiency of synthetic SL agonists in triggering signaling through the Striga SL receptor, ShHTL7, depends on the receptor-catalyzed hydrolytic reaction of the agonists.
[more...]
Use: pFind



Characterization of protein unfolding by fast cross-linking mass spectrometry using di-ortho-phthalaldehyde cross-linkers
Nature communications. 2022. Wang, JH et al. Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Peking Univ, Coll Chem & Mol Engn, Peking Tsinghua Ctr Life Sci,Minist Educ, Beijing Natl Lab Mol Sci,Key Lab Bioorgan Chem &, Beijing 100871, Peoples R China; Chinese Acad Sci, Innovat Acad Precis Measurement Sci & Technol, Wuhan 430071, Peoples R China
ABSTRACT: Conformations sampled by a protein while it unfolds are difficult to visualize. Here, the authors develop di-ortho-phthalaldehyde cross-linkers for rapid chemical cross-linking mass spectrometry analysis and demonstrate that this method captures the conformations of protein unfolding intermediates.Chemical cross-linking of proteins coupled with mass spectrometry is widely used in protein structural analysis. In this study we develop a class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is called DOPA2.
[more...]
Use: pFind; pLink



Deep coverage proteome analysis of hair shaft for forensic individual identification
Forensic Science International: Genetics. 2022. Wu, JL et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China; Peoples Publ Secur Univ China, Grad Sch, 1 Muxidi Nanli, Beijing 100038, Peoples R China; Inst Forens Sci, Natl Engn Lab Forens Sci, Key Lab Forens Genet, Minist Publ Secur, 17 Muxidi Nanli, Beijing 100038, Peoples R China
ABSTRACT: Hair shaft is one of the most common biological evidence found at crime scenes. However, due to the biogenic degradation of nuclear DNA in hair shaft, it is difficult to achieve individual identification through routine DNA analysis. In contrast, the proteins in hair shaft are stable and contain genetic polymorphisms in the form of single amino acid polymorphisms (SAPs), translated from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome. However, the number of SAPs detected still cannot meet the requirements of practical applications.
[more...]
Use: pFind



Efficient detection of the alternative spliced human proteome using translatome sequencing
Frontiers in Molecular Biosciences. 2022. Wu, Chun et al. Jinan Univ, Inst Life & Hlth Engn, MOE Key Lab Tumor Mol Biol, Guangzhou, Peoples R China; Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Guangzhou, Peoples R China
ABSTRACT: Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms.
[more...]
Use: pFind



Comprehensive identification of protein orthologs in the family Ascoviridae facilitates an understanding of phylogenomics, protein conservation, and phosphorylation
Archives of Virology. 2022. Shi, YH et al. Weifang Med Univ, Sch Life Sci & Technol, Weifang 261053, Peoples R China; Hunan Agr Univ, Hunan Prov Key Lab Biol & Control Plant Dis & Ins, Changsha 410128, Hunan, Peoples R China
ABSTRACT: Analysis of orthology is important for understanding protein conservation, function, and phylogenomics. In this study, we performed a comprehensive analysis of gene orthology in the family Ascoviridae based on identification of 366 protein homologue groups and phylogenetic analysis of 34 non-single-copy proteins. Our findings revealed 90 newly annotated proteins, five newly identified core proteins for the family Ascoviridae, and 14 core proteins for the genus Ascovirus. A phylogenomic tree of 11 Ascoviridae members was constructed based on a concatenation of 35 of the 45 ortholog groups.
[more...]
Use: pFind



A mechanism for oxidative damage repair at gene regulatory elements
Nature. 2022. Ray, S et al. Univ Sheffield, Hlth Lifespan & Neurosci Inst, Sheffield, S Yorkshire, England; Univ Bradford, Sch Pharm & Med Sci, Inst Canc Therapeut, Bradford, W Yorkshire, England; Univ Sheffield, Sch Biosci, Sheffield, S Yorkshire, England
ABSTRACT: Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation(1,2). Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1.
[more...]
Use: pFind



Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme
BIOCHEMISTRY. 2022. Shivakumaraswamy, S et al. Jawaharlal Nehru Ctr Adv Sci Res, Mol Biol & Genet Unit, Bengaluru 560064, India
ABSTRACT: Guanosine 5 & PRIME;-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 & PRIME;-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP.
[more...]
Use: pFind; pLink



Itaconate and itaconate derivatives target JAK1 to suppress alternative activation of macrophages
Cell Metabolism. 2022. Runtsch, MC et al. Trinity Coll Dublin, Sch Biochem & Immunol, Trinity Biomed Sci Inst, 152-160 Pearse St, Dublin D02 R590, Ireland
ABSTRACT: The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory "M1" macrophages. However, alternatively activated "M2" macrophages can take up itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI.
[more...]
Use: pFind



AlphaPeptDeep: a modular deep learning framework to predict peptide properties for proteomics
Nature Communications. 2022. Zeng, Wen-Feng et al. Univ Copenhagen, Fac Hlth Sci, NNF Ctr Prot Res, Prote Program, Copenhagen, Denmark; Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany
ABSTRACT: Machine learning and in particular deep learning (DL) are increasingly important in mass spectrometry (MS)-based proteomics. Recent DL models can predict the retention time, ion mobility and fragment intensities of a peptide just from the amino acid sequence with good accuracy. However, DL is a very rapidly developing field with new neural network architectures frequently appearing, which are challenging to incorporate for proteomics researchers. Here we introduce AlphaPeptDeep, a modular Python framework built on the PyTorch DL library that learns and predicts the properties of peptides (https://github.com/MannLabs/alphapeptdeep).
[more...]
Use: pFind; pDeep



Mucus sialylation determines intestinal host-commensal homeostasis
Cell. 2022. Yao, YK et al. NIAID, Mol Dev Immune Syst Sect, Lab Immune Syst Biol & Clin Genom Program, NIH, Bethesda, MD 20892 USA; NCI, Expt Immunol Branch, NIH, Bethesda, MD 20892 USA
ABSTRACT: Intestinal mucus forms the first line of defense against bacterial invasion while providing nutrition to support microbial symbiosis. How the host controls mucus barrier integrity and commensalism is unclear. We show that terminal sialylation of glycans on intestinal mucus by ST6GALNAC1 (ST6), the dominant sialyltransferase specifically expressed in goblet cells and induced by microbial pathogen-associated molecular patterns, is essential for mucus integrity and protecting against excessive bacterial proteolytic degradation.
[more...]
Use: pFind



HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors
Nature communications. 2022. Guan, L et al. Univ Penn, Sch Arts & Sci, Dept Biol, Philadelphia, PA 19104 USA
ABSTRACT: The lack of tumor infiltration by CD8(+) T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8(+) T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8(+) T cells into tumors.
[more...]
Use: pFind; pLink



Glyco-Decipher enables glycan database-independent peptide matching and in-depth characterization of site-specific N-glycosylation
Nature Communications. 2022. Fang, Z et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Dalian Univ Technol, Sch Bioengn, Dalian 116024, Peoples R China
ABSTRACT: Glycopeptides with unusual glycans or poor peptide backbone fragmentation in tandem mass spectrometry are unaccounted for in typical site-specific glycoproteomics analysis and thus remain unidentified. Here, we develop a glycoproteomics tool, Glyco-Decipher, to address these issues. Glyco-Decipher conducts glycan database-independent peptide matching and exploits the fragmentation pattern of shared peptide backbones in glycopeptides to improve the spectrum interpretation. We benchmark Glyco-Decipher on several large-scale datasets, demonstrating that it identifies more peptide-spectrum matches than Byonic, MSFragger-Glyco, StrucGP and pGlyco 3.0, with a 33.5%-178.5% increase in the number of identified glycopeptide spectra.
[more...]
Use: pFind; pGlyco; pQuant



Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state
Nature Structural & Molecular Biology. 2022. Cai, Sarah W. et al. Rockefeller Univ, Lab Mol Electron Microscopy, New York, NY 10065 USA; Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA
ABSTRACT: The CST-Pol alpha/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-angstrom resolution cryo-EM structure of human CST-Pol alpha/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus.
[more...]
Use: pFind; pLink



Crucial role and mechanism of transcription-coupled DNA repair in bacteria
Nature. 2022. Bharati, BK et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10012 USA; NYU, Sch Med, Howard Hughes Med Inst, New York, NY 10012 USA; Chinese Acad Sci, Inst Plant Physiol & Ecol, Ctr Excellence Mol Plant Sci, Key Lab Synthet Biol, Shanghai, Peoples R China
ABSTRACT: Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype(1-3). Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo.
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Shelterin is a dimeric complex with extensive structural heterogeneity
PNAS. 2022. Zinder, John C. et al. Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA; Rockefeller Univ, Lab Mol Elect Microscopy, New York, NY 10065 USA
ABSTRACT: Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits.
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Histone deacetylase 3 contributes to the antiviral innate immunity of macrophages by interacting with FOXK1 to regulate STAT1/2 transcription
Cell Reports. 2022. Yang, LP et al. Zhejiang Univ, Affiliated Hosp 2, Dept Infect Dis, Sch Med, Hangzhou 310009, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Syst Med, Beijing 100005, Peoples R China; Suzhou Inst Syst Med, Suzhou 215123, Peoples R China; Univ Calif Los Angeles, Dept Microbiol Immunol & Mol Genet, Los Angeles, CA 90095 USA
ABSTRACT: It is well known that interferon (IFN)-alpha/-beta activates the JAK/STAT signaling pathway and suppresses viral replication through the induction of IFN stimulated genes (ISGs). Here, we report that knockout of HDAC3 from macrophages results in the decreased expression of STAT1 and STAT2, leading to defective antiviral immunity in cells and mice. Further studies show that HDAC3 interacts with a conserved transcription factor Forkhead Box K1 (FOXK1), co-localizes with FOXK1 at the promoter of STAT1 and STAT2, and is required for protecting FOXK1 from lysosomal system-mediated degradation, FOXK1 -deficient macrophages also show low STAT1 and STAT2 expression with defective responses to viruses.
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Protein posttranslational signatures identified in COVID-19 patient plasma
Frontiers in Cell and Developmental Biology. 2022. Vedula, Pavan et al. Univ Penn, Sch Vet Med, Dept Biomed Sci, Philadelphia, PA 19104 USA
ABSTRACT: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease.
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Iso-seco-tanapartholide activates Nrf2 signaling pathway through Keap1 modification and oligomerization to exert anti-inflammatory effects
Free Radical Biology and Medicine. 2022. Zhu, DR et al. China Pharmaceut Univ, Nanjing 210009, Peoples R China
ABSTRACT: Covalent modification of Keap1 results in reducing ubiquitination and the accumulation of Nrf2, which subsequently initiates the transcription of cellular anti-oxidant and anti-inflammatory genes. Iso-seco-tanapartholide (IST), a sesquiterpene isolated from the traditional Chinese medicine Artemisia argyi, had been reported to possess NF-Kappa B inhibitory activity. However, its deep anti-inflammatory effects and direct target have never been reported. Here we show that IST activated Nrf2 and increased its target gene expression.
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Orally efficacious lead of the AVG inhibitor series targeting a dynamic interface in the respiratory syncytial virus polymerase
Science Advances. 2022. Sourimant, J et al. Georgia State Univ, Ctr Translat Antiviral Res, Inst Biomed Sci, Atlanta, GA 30303 USA
ABSTRACT: Respiratory syncytial virus (RSV) is a leading cause of lower respiratory infections in infants and the immuno-compromised, yet no efficient therapeutic exists. We have identified the AVG class of allosteric inhibitors of RSV RNA synthesis. Here, we demonstrate through biolayer interferometry and in vitro RNA-dependent RNA polymerase (RdRP) assays that AVG compounds bind to the viral polymerase, stalling the polymerase in initiation conformation. Resistance profiling revealed a unique escape pattern, suggesting a discrete docking pose.
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Deephos: predicted spectral database search for TMT-labeled phosphopeptides and its false discovery rate estimation
Bioinformatics. 2022. Na, S et al. Hanyang Univ, Inst Artificial Intelligence Res, Seoul 04763, South Korea; Hanyang Univ, Dept Comp Sci, Seoul 04763, South Korea
ABSTRACT: Motivation: Tandem mass tag (TMT)-based tandem mass spectrometry (MS/MS) has become the method of choice for the quantification of post-translational modifications in complex mixtures. Many cancer proteogenomic studies have highlighted the importance of large-scale phosphopeptide quantification coupled with TMT labeling. Herein, we propose a predicted Spectral DataBase (pSDB) search strategy called Deephos that can improve both sensitivity and specificity in identifying MS/MS spectra of TMT-labeled phosphopeptides.Results: With deep learning-based fragment ion prediction, we compiled a pSDB of TMT-labeled phosphopeptides generated from similar to 8000 human phosphoproteins annotated in UniProt.
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AlphaFold and structural mass spectrometry enable interrogations on the intrinsically disordered regions in cyanobacterial light-harvesting complex phycobilisome
JOURNAL OF MOLECULAR BIOLOGY. 2022. Liu, Haijun et al. Washington Univ St Louis, Dept Biol, One Brookings Dr,POB 1137, St Louis, MO 63130 USA
ABSTRACT: Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of pro-teins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for inter-rogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS).
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Identification of microproteins in Hep3B cells at different cell cycle stages
Journal of Proteome Research. 2022. Li, B et al. Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Hubei, Peoples R China; Cent China Normal Univ, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China
ABSTRACT: Microproteins are generated from small open reading frames andturn out to play various vital biological functions. As an essential biological event ofeukaryotic cells, the cell cycle is involved in cell replication and division. For such ahighly regulated event, microproteins associated with cell cycle regulation remainedunclarified. Utilizing a combination of bottom-up and top-down proteomics, weanalyzed microproteins at specific cell cycle stages of Hep3B cells. A total of 657microproteins were identified under three cell cycle stages, including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the G2/M stage.
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EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1α is repressed by SET7-catalyzed lysine methylation
Journal of Biological Chemistry. 2022. Tang, JH et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Innovat Seed Design, Wuhan, Peoples R China; Hubei Hongshan Lab, Wuhan, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China
ABSTRACT: Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 alpha under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 alpha.
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Targeting tumor endothelial hyperglycolysis enhances immunotherapy through remodeling tumor microenvironment
Acta Pharmaceutica Sinica B. 2022. Shan, YL et al. China Pharmaceut Univ, Key Lab Drug Metab & Pharmacokinet, State Key Lab Nat Med, Nanjing 210009, Peoples R China; Nanjing Med Univ, Dept Hematol & Oncol, Affiliated Huaian 1 Peoples Hosp, Huaian 223300, Peoples R China
ABSTRACT: Vascular abnormality isa hallmark of most solid tumors and facilitates immune evasion. Targeting the abnormal metabolism of tumor endothelial cells (TECs) may provide an opportunity to improve the outcome of immunotherapy. Here, in comparison to vascular endothelial cells from adjacent peritumoral tissues in patients with colorectal cancer (CRC), TECs presented enhanced glycolysis with higher glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Then an unbiased screening identified that osimertinib could modify the GAPDH and thus inhibit its activity in TECs.
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Accurate discrimination of leucine and isoleucine residues by combining continuous digestion with multiple MS3 spectra integration in protein sequence
Talanta. 2022. Zhang, Weijie et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: Protein de novo sequencing based on tandem mass spectrometry is a crucial technology that enables the identification of peptides without searching databases and assembling unknown sequence proteins, especially for monoclonal antibodies (mAbs). However, the discrimination of leucine (Leu) and isoleucine (Ile) residues in the target protein sequence is still challenging. Herein, we developed an accurate method by continuous digestion with MS3-based fragmentation and multiple spectra integration (evaluated by combined verification score, CVS) to distinguish Leu and Ile residues.
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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic …
FRONTIERS IN MICROBIOLOGY. 2022. Songhao Jiang et al. Guangzhou Univ Chinese Med, Clin Med Coll 2, Guangzhou Higher Educ Mega Ctr, Guangzhou, Peoples R China; Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Prote,Res, Beijing, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Med Biotechnol, Res Unit Prote & Res & Dev New Drug, Beijing, Peoples R China; Hebei Univ, Sch Life Sci, Key Lab Microbial Divers Res & Applicat Hebei, Baoding, Peoples R China
ABSTRACT: Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis.
[more...]
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Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes
Genomics. 2022. Shi, JH et al. Chinese Acad Med Sci, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Proteom, Res Unit Prote & Res & Dev New Drug,Inst Lifeom, Beijing 102206, Peoples R China
ABSTRACT: Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5 ' end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes.
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Ac-LysargiNase efficiently helps genome reannotation of Mycolicibacterium smegmatis MC2 155
Journal of Proteomics. 2022. Zhu, HM et al. Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr,State Key Lab Prote, Natl Ctr Prot Sci Beijing,Res Unit Prote & Res &, Beijing 102206, Peoples R China
ABSTRACT: Accurate genome annotation, the foundation of life science research in the genome era, is hampered by limited known gene models, nonstandard start codons, and the limited homology of annotated genes in other organisms. LysargiNase mirrors trypsin at the cleavage sites, providing the opportunity to identify peptides other than tryptic peptides. In this study, we used an in-house developed acetylated LysargiNase (Ac-LysargiNase) with higher activity and stability in non-pathogenic Mycolicibacterium smegmatis MC2 155 to supplement the widely used trypsin in proteomic studies.
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A novel proteogenomic integration strategy expands the breadth of neo-epitope sources
Cancers. 2022. Xiang, Haitao et al. BGI Shenzhen, Shenzhen 518103, Peoples R China; Guangdong Prov Key Lab Human Dis Genom, Shenzhen Key Lab Genom, Shenzhen 518083, Peoples R China; BGI, Shenzhen 518083, Peoples R China
ABSTRACT: Simple Summary Tumor-specific antigens are ideal targets for cancer immunotherapy. Mass spectrometry, which is the main method that directly identifies neo-epitopes presented on tumor cells, focuses mainly on peptides derived from annotated protein-coding exomes. However, non-canonical peptides arising from alterations at genomic, transcriptional, and posttranslational levels have been identified in several pioneering studies, making it necessary to develop an integrated proteogenomic approach that can comprehensively identify neoantigens derived from all genomic regions.
[more...]
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De-sialylation of glycopeptides by acid treatment: enhancing sialic acid removal without reducing the identification
Analytical Methods. 2022. Dong, Wenbo et al. Northwest Univ, Coll Life Sci, Xian 710069, Shaanxi, Peoples R China
ABSTRACT: Sialic acid, a common terminal monosaccharide on many glycoconjugates, plays essential roles in many biological processes such as immune responses, pathogen recognition, and cancer development. For various purposes, sialic acids may need to be removed from glycopeptides or glycans, mainly using enzymatical or chemical approaches. In this study, we found that most commonly used chemical methods couldn't completely remove sialic acids from glycopeptides. Although the de-sialylation efficiency could be further enhanced by increasing the treatment time or acid concentration, the undesirable side reactions on the peptide portion would decrease glycopeptide identification.
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Quantitative model suggests both intrinsic and contextual features contribute to the transcript coding ability determination in cells
Briefings in Bioinformatics. 2022. Kang, Yu-Jian et al. Peking Univ, Biomed Pioneering Innovat Ctr BIOPIC, Beijing Adv Innovat Ctr Genom ICG, Ctr Bioinformat CBI,Sch Life Sci, Beijing 100871, Peoples R China; Peking Univ, State Key Lab Pry Lein & Plant Gene Res, Sch Life Sci, Beijing 100871, Peoples R China
ABSTRACT: Gene transcription and protein translation are two key steps of the 'central dogma.' It is still a major challenge to quantitatively deconvolute factors contributing to the coding ability of transcripts in mammals. Here, we propose ribosome calculator (RiboCalc) for quantitatively modeling the coding ability of RNAs in human genome. In addition to effectively predicting the experimentally confirmed coding abundance via sequence and transcription features with high accuracy, RiboCalc provides interpretable parameters with biological information.
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Angiotensin-converting enzyme genotype--specific immune response contributes to the susceptibility of COVID-19: a nested case--control study
Frontiers in pharmacology. 2022. Gong, Pengyun et al. Hubei Ctr Dis Control & Prevent, Wuhan, Peoples R China; Beihang Univ, Sch Biol Sci & Med Engn, Beijing, Peoples R China; Capital Med Univ, Beijing YouAn Hosp, Dept Radiol, Beijing, Peoples R China; Beihang Univ, Sch Engn Med, Beijing, Peoples R China
ABSTRACT: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has resulted in a global pandemic.Methodology: We used a two-step polymerase chain reaction to detect the ACE genotype and ELISA kits to detect the cytokine factor. We also used proteomics to identify the immune pathway related to the ACE protein expression.Result: In this study, we found that the angiotensin-converting enzyme (ACE) deletion polymorphism was associated with the susceptibility to COVID-19 in a risk-dependent manner among the Chinese population.
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Discovery of 194 Unreported Conopeptides and Identification of a New Protein Disulfide Isomerase in Conus caracteristicus Using Integrated Transcriptomic and Proteomic Analysis
Frontiers in Marine Science. 2022. Zhang, Han et al. Southern Med Univ, Guangdong Prov Key Lab Single Cell Technol & Appl, Guangzhou, Peoples R China; Southern Med Univ, Guangdong Hong Kong Macao Greater Bay Area Ctr Br, Hong Kong, Guangdong, Peoples R China; Southern Med Univ, Sch Basic Med Sci, Dept Biochem & Mol Biol, Guangzhou, Peoples R China
ABSTRACT: Current ConoServer database accumulates 8,134 conopeptides from 122 species of cone snail, which are pharmaceutically attractive marine resource. However, many more conopeptides remain to be discovered, and the enzymes involved in their synthesis and processing are unclear. In this report, firstly we screened and analyzed the differentially expressed genes (DEGs) between venom duct (VD) and venom bulb (VB) of C. caracteristicus, and obtained 3,289 transcripts using a comprehensive assembly strategy.
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MetaLab-MAG: A Metaproteomic Data Analysis Platform for Genome-Level Characterization of Microbiomes from the Metagenome-Assembled Genomes Database
Journal of Proteome Research. 2022. Cheng, Kai et al. Univ Ottawa, Fac Med, Sch Pharmaceut Sci, Ottawa, ON K1H 8M5, Canada
ABSTRACT: The studies of microbial communities have drawn increased attention in various research fields such as agriculture, environment, and human health. Recently, metaproteomics has become a powerful tool to interpret the roles of the community members by investigating the expressed proteins of the microbes. However, analyzing the metaproteomic data sets at genome resolution is still challenging because of the lack of efficient bioinformatics tools. Here we develop MetaLab-MAG, a specially designed tool for the characterization of microbiomes from metagenome-assembled genomes databases.
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Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes
CELL. 2022. Jin, Zeyu et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou 310024, Zhejiang, Peoples R China
ABSTRACT: The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-en-coded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercom-plex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid b-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110.
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Assessing the dark field of metaproteome
Analytical Chemistry. 2022. Duan, Haonan et al. Univ Ottawa, Fac Med, Daniel Figeys Sch Pharmaceut Sci, Ottawa, ON K1H 8L1, Canada; Univ Ottawa, Ottawa Inst Syst Biol, Ottawa, ON K1H 8L1, Canada
ABSTRACT: The human gut microbiome is a complex system composed of hundreds of species, and metaproteomics can be used to explore their expressed functions. However, many lower abundance species are not detected by current metaproteomic techniques and represent the dark field of metaproteomics. We do not know the minimal abundance of a bacterium in a microbiome-(depth) that can be detected by shotgun metaproteomics. In this study, we spiked 15N-labeled E. coli peptides at different percentages into peptides mixture derived from the human gut microbiome to evaluate the depth that can be achieved by shotgun metaproteomics.
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Changes to Urinary Proteome in High-Fat-Diet ApoE-/- Mice
Biomolecules. 2022. Hua, Yuanrui et al. Beijing Normal Univ, Coll Life Sci, Gene Engn Drug & Biotechnol Beijing Key Lab, Beijing 100875, Peoples R China
ABSTRACT: Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points.
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The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay
NATURE PLANTS. 2022. Bi, Guozhi et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing, Peoples R China; Hainan Yazhou Bay Seed Lab, Sanya, Peoples R China; Univ Chinese Acad Sci, Coll Adv Agr Sci, Beijing, Peoples R China; Univ Chinese Acad Sci, CAS Ctr Excellence Biot Interact, Beijing, Peoples R China; Beijing Inst Life, Natl Ctr Prot Sci, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing, Peoples R China
ABSTRACT: Rapid production of H2O2 is a hallmark of plant responses to diverse pathogens and plays a crucial role in signalling downstream of various receptors that perceive immunogenic patterns. However, mechanisms by which plants sense H2O2 to regulate immunity remain poorly understood. We show that endogenous H2O2 generated upon immune activation is sensed by the thiol peroxidase PRXIIB via oxidation at Cys51, and this is essential for stomatal immunity against Pseudomonas syringae. We further show that in immune-stimulated cells, PRXIIB conjugates via Cys51 with the type 2C protein phosphatase ABA insensitive 2 (ABI2), subsequently transducing H2O2 signal to ABI2.
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PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling
Frontiers in Cell and Developmental Biology. 2022. Zhai, Linhui et al. Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Shanghai, Peoples R China; Nanjing Univ Chinese Med, Sch Pharm, Jiangsu Key Lab Funct Subst Chinese Med, Nanjing, Jiangsu, Peoples R China; Nanjing Univ Chinese Med, Sch Chinese Mat Med, Sch Pharm, Nanjing, Jiangsu, Peoples R China
ABSTRACT: High-throughput profiling of protein C-termini is still a challenging task. Proteomics provides a powerful technology for systematic and high-throughput study of protein C-termini. Various C-terminal peptide enrichment strategies based on chemical derivatization and chromatography separation have been reported. However, they are still costly and time-consuming, with low enrichment efficiency for C-terminal peptides. In this study, by taking advantage of the high reaction selectivity of 2-pyridinecarboxaldehyde (2-PCA) with an alpha-amino group on peptide N-terminus and high affinity between biotin and streptavidin, we developed a 2-PCA- and biotin labeling-based C-terminomic (PBC) strategy for a high-efficiency and high-throughput analysis of protein C-terminome.
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Many kinds of oxidized proteins are present more in the urine of the elderly
Clinical proteomics. 2022. Liu, YT et al. Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing 100875, Peoples R China
ABSTRACT: Background Many studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages. Methods Discovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications.
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PML Body Component Sp100A Is a Cytosolic Responder to IFN and Activator of Antiviral ISGs
Mbio. 2022. Dong, HC et al. Sun Yat Sen Univ, Key Lab Trop Dis Control, Minist Educ, Guangzhou, Peoples R China; Sun Yat Sen Univ, Ctr Infect & Immun Studies, Sch Med, Shenzhen, Peoples R China
ABSTRACT: PML bodies sit at the center stage of various important biological processes; however, the signal transduction networks of these macromolecular protein complexes remain enigmatic. The present study illustrates, in detail and for the first time, the course of signal receiving, processing, and implementation by PML bodies in response to IFN and virus infection.Promyelocytic leukemia protein (PML) bodies are implicated in one of the key pathways in the establishment of antiviral status in response to interferon (IFN), yet the molecular mechanisms bridging the cross talk remain elusive.
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The effect of lactulose thermal degradation products on $\beta$-lactoglobulin: Linear-, loop-, and cross-link structural modifications and reduced digestibility
FOOD CHEMISTRY. 2022. Dong, L et al. Nankai Univ, Sch Med, Tianjin Key Lab Food Sci & Hlth, Tianjin 300071, Peoples R China
ABSTRACT: The thermal degradation products of lactulose and the interaction between lactulose and beta-lactoglobulin (beta Lg) were investigated in a thermal model system. Lactulose was thermally degraded into fructose and galactose, which were further degraded into methylglyoxal, glyoxal, 3-deoxyglucosone, and 2, 3-butanedione via heating. After incubating with lactulose, the structure of beta Lg was changed, which manifested by the formation of new band with doubled the molecular weight of beta Lg in the mobility spectrum and the changes in the internal fluo-rescence spectrum.
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FBB18 participates in preassembly of almost all axonemal dyneins independent of R2TP complex
PLoS genetics. 2022. Wang, LM et al. Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Shandong, Peoples R China; Tsinghua Univ, Sch Life Sci, MOE Key Lab Prot Sci, Beijing, Peoples R China
ABSTRACT: Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms.
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Cryo-EM structure of the human CST--Pol$\alpha$/primase complex in a recruitment state
Nature Structural & Molecular Biology. 2022. Cai, Sarah W. et al. Rockefeller Univ, Lab Mol Electron Microscopy, New York, NY 10065 USA; Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA
ABSTRACT: The CST-Pol alpha/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-angstrom resolution cryo-EM structure of human CST-Pol alpha/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus.
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Mirror proteases of Ac-Trypsin and Ac-LysargiNase precisely improve novel event identifications in Mycolicibacterium smegmatis MC2 155 by proteogenomic analysis
Frontiers in Microbiology. 2022. Jiang, Songhao et al. Hebei Univ, Sch Life Sci, Key Lab Microbial Divers Res & Applicat Hebei, Baoding, Peoples R China; Guangzhou Univ Chinese Med, Clin Med Coll 2, Guangzhou Higher Educ Mega Ctr, Guangzhou, Peoples R China; Chinese Acad Med Sci, Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Prote,Res, Beijing, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Inst Med Biotechnol, Res Unit Prote & Res & Dev New Drug, Beijing, Peoples R China
ABSTRACT: Accurate identification of novel peptides remains challenging because of the lack of evaluation criteria in large-scale proteogenomic studies. Mirror proteases of trypsin and lysargiNase can generate complementary b/y ion series, providing the opportunity to efficiently assess authentic novel peptides in experiments other than filter potential targets by different false discovery rates (FDRs) ranking. In this study, a pair of in-house developed acetylated mirror proteases, Ac-Trypsin and Ac-LysargiNase, were used in Mycolicibacterium smegmatis MC2 155 for proteogenomic analysis.
[more...]
Use: pFind



A Novel Proteogenomic Integration Strategy Expands the Breadth of Neo-Epitope Sources
Cancers. 2022. Xiang, Haitao et al. BGI Shenzhen, Shenzhen 518103, Peoples R China; Guangdong Prov Key Lab Human Dis Genom, Shenzhen Key Lab Genom, Shenzhen 518083, Peoples R China; BGI, Shenzhen 518083, Peoples R China
ABSTRACT: Simple Summary Tumor-specific antigens are ideal targets for cancer immunotherapy. Mass spectrometry, which is the main method that directly identifies neo-epitopes presented on tumor cells, focuses mainly on peptides derived from annotated protein-coding exomes. However, non-canonical peptides arising from alterations at genomic, transcriptional, and posttranslational levels have been identified in several pioneering studies, making it necessary to develop an integrated proteogenomic approach that can comprehensively identify neoantigens derived from all genomic regions.
[more...]
Use: pFind



The effect of lactulose thermal degradation products on β-lactoglobulin: linear-, loop-, and cross-link structural modifications and reduced digestibility
FOOD CHEMISTRY. 2022. Dong, L et al. Nankai Univ, Sch Med, Tianjin Key Lab Food Sci & Hlth, Tianjin 300071, Peoples R China
ABSTRACT: The thermal degradation products of lactulose and the interaction between lactulose and beta-lactoglobulin (beta Lg) were investigated in a thermal model system. Lactulose was thermally degraded into fructose and galactose, which were further degraded into methylglyoxal, glyoxal, 3-deoxyglucosone, and 2, 3-butanedione via heating. After incubating with lactulose, the structure of beta Lg was changed, which manifested by the formation of new band with doubled the molecular weight of beta Lg in the mobility spectrum and the changes in the internal fluo-rescence spectrum.
[more...]
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EGLN1 prolyl hydroxylation of hypoxia-induced transcription factor HIF1$\alpha$ is repressed by SET7-catalyzed lysine methylation
Journal of Biological Chemistry. 2022. Tang, JH et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Innovat Seed Design, Wuhan, Peoples R China; Hubei Hongshan Lab, Wuhan, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China
ABSTRACT: Egg-laying defective nine 1 (EGLN1) functions as an oxygen sensor to catalyze prolyl hydroxylation of the transcription factor hypoxia-inducible factor-1 alpha under normoxia conditions, leading to its proteasomal degradation. Thus, EGLN1 plays a central role in the hypoxia-inducible factor-mediated hypoxia signaling pathway; however, the posttranslational modifications that control EGLN1 function remain largely unknown. Here, we identified that a lysine monomethylase, SET7, catalyzes EGLN1 methylation on lysine 297, resulting in the repression of EGLN1 activity in catalyzing prolyl hydroxylation of hypoxia-inducible factor-1 alpha.
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Identification of Microproteins in Hep3B Cells at Different Cell Cycle Stages
Journal of Proteome Research. 2022. Li, B et al. Cent China Normal Univ, Sch Life Sci, Wuhan 430079, Hubei, Peoples R China; Cent China Normal Univ, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China
ABSTRACT: Microproteins are generated from small open reading frames andturn out to play various vital biological functions. As an essential biological event ofeukaryotic cells, the cell cycle is involved in cell replication and division. For such ahighly regulated event, microproteins associated with cell cycle regulation remainedunclarified. Utilizing a combination of bottom-up and top-down proteomics, weanalyzed microproteins at specific cell cycle stages of Hep3B cells. A total of 657microproteins were identified under three cell cycle stages, including 151 in the G0/G1 stage, 163 in the S stage, and 132 in the G2/M stage.
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Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity
PNAS. 2022. Zinder, John C. et al. Rockefeller Univ, Lab Cell Biol & Genet, New York, NY 10065 USA; Rockefeller Univ, Lab Mol Elect Microscopy, New York, NY 10065 USA
ABSTRACT: Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits.
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Nitrogen mustard alkylates and cross-links p53 in human keratinocytes
Chemical Research in Toxicology. 2022. Jan, YH et al. Rutgers State Univ, Sch Publ Hlth, Dept Environm & Occupat Hlth & Justice, Piscataway, NJ 08854 USA
ABSTRACT: Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress.
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Subcellular interactomes revealed by merging APEX with cross-linking mass spectrometry
Analytical Chemistry. 2022. Sun, MZ et al. Chinese Inst Brain Res CIBR, Beijing 102206, Peoples R China; Peking Univ, Synthet & Funct Biomol Ctr, Coll Chem & Mol Engn, Dept Chem Biol,Beijing Natl Lab Mol Sci,Key Lab Bi, Beijing 100871, Peoples R China; Inst Canc Res, Shenzhen Bay Lab, Shenzhen 518107, Peoples R China; Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, PKU IDG McGovern Inst Brain Res, Beijing 100871, Peoples R China
ABSTRACT: Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation.
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Alkynyl-enrichable carboxyl-selective crosslinkers to increase the crosslinking coverage for deciphering protein structures
Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N- hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples.
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Structure of human chromatin-remodelling PBAF complex bound to a nucleosome
Nature. 2022. Yuan, JJ et al. Tsinghua Univ, MOE Key Lab Prot Sci, Beijing, Peoples R China; Tsinghua Univ, Sch Life Sci, Beijing, Peoples R China; Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Beijing, Peoples R China
ABSTRACT: DNA wraps around the histone octamer to form nucleosomes(1), the repeating unit of chromatin, which create barriers for accessing genetic information. Snf2-like chromatin remodellers couple the energy of ATP binding and hydrolysis to reposition and recompose the nucleosome, and have vital roles in various chromatin-based transactions(2,3). Here we report the cryo-electron microscopy structure of the 12-subunit human chromatin-remodelling polybromo-associated BRG1-associated factor (PBAF) complex bound to the nucleosome.
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Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II
Nature Structural & Molecular Biology. 2022. Rengachari, S et al. Inst Canc Res, Div Struct Biol, London, England; Human Technopole, Milan, Italy; Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany
ABSTRACT: Rengachari et al. provide a structural investigation of Pol II initiation at snRNA gene promoters and find that the snRNA-activating protein complex enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro.RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level.
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Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors
PNAS. 2022. Su, JY et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, State Key Lab Membrane Biol,Sch Life Sci, Beijing 100084, Peoples R China
ABSTRACT: Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive.
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Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly
Nature communications. 2022. Fischer, ES et al. MRC Lab Mol Biol, Cambridge Biomed Campus,Francis Crick Ave, Cambridge CB2 0QH, England
ABSTRACT: Formation of the mitotic checkpoint complex (MCC) is catalysed by a phosphorylation-dependent scaffold. This work provides structural details of how a tripartite Mad1:Bub1:Cdc20 complex presents Cdc20 to Mad2, triggering open-to-closed conversion of Mad2 to assemble the MCC.In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase-promoting complex/cyclosome, thereby delaying entry into anaphase.
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Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH
Nature Plants. 2022. Yu, GM et al. Umea Univ, Dept Plant Physiol, Umea Plant Sci Ctr UPSC, Umea, Sweden; Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Natl Lab Biomacromol, Beijing, Peoples R China
ABSTRACT: Genetic, biochemical and high-resolution structural studies of chloroplast protein SOQ1 reveal the existence of a C-terminal lumenal domain with potential redox function and its essential role for suppressing photoprotection in plants.Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP.
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Identification of an autoinhibitory, mitophagy-inducing peptide derived from the transmembrane domain of USP30
Autophagy. 2022. Qin, X et al. Pingshan Translat Med Ctr, Shenzhen Bay Lab, Shenzhen, Peoples R China; Peking Univ, Sch Chem Biol & Biotechnol, State Key Lab Chem Oncogen, Shenzhen Grad Sch, Shenzhen, Peoples R China; Tsinghua Univ, Tsinghua Shenzhen Int Grad Sch, Inst Biopharmaceut & Hlth Engn, Shenzhen, Peoples R China
ABSTRACT: The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide.
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Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation
Nature communications. 2022. Levina, A et al. Max Perutz Labs, Dept Struct & Computat Biol, Campus Vienna Bioctr 5, A-1030 Vienna, Austria; Med Univ Vienna, Dept Med Biochem, A-1090 Vienna, Austria
ABSTRACT: 3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP3-mediated face-to-face dimer.
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Conformational rearrangements upon start codon recognition in human 48S translation initiation complex
Nucleic Acids Research. 2022. Yi, Sung-Hui et al. Max Planck Inst Multidisciplinary Sci, Dept Struct Dynam, D-37077 Gottingen, Germany; Georg August Univ Gottingen, Inst Microbiol & Genet, Dept Mol Struct Biol, D-37077 Gottingen, Germany
ABSTRACT: Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNA(i)(Met) and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step.
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In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker
Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian Inst Chem Phys, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference.
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Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows
Nature Communications. 2022. Matzinger, Manuel et al. Austrian Acad Sci, Vienna BioCtr VBC, Inst Mol Biotechnol, Vienna, Austria; Vienna BioCtr VBC, Inst Mol Pathol IMP, Vienna, Austria
ABSTRACT: Cross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows.
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Structure of nucleosome-bound human PBAF complex
Nature Communications. 2022. Wang, Li et al. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, State Key Laboratory of Genetic Engineering and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College of Fudan University, Shanghai, 200032, China The International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, China, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, China Human Phenome Institute, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, 200433, China
ABSTRACT: BAF and PBAF are mammalian SWI/SNF family chromatin remodeling complexes that possess multiple histone/DNA-binding subunits and create nucleosome-depleted/free regions for transcription activation. Despite previous structural studies and recent advance of SWI/SNF family complexes, it remains incompletely understood how PBAF-nucleosome complex is organized. Here we determined structure of 13-subunit human PBAF in complex with acetylated nucleosome in ADP-BeF3-bound state. Four PBAF-specific subunits work together with nine BAF/PBAF-shared subunits to generate PBAF-specific modular organization, distinct from that of BAF at various regions.
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Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome
PNAS. 2022. Rahman, S et al. Johns Hopkins Univ, Dept Biophys & Biophys Chem, Sch Med, Baltimore, MD 21205 USA
ABSTRACT: The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes.
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Hsp multichaperone complex buffers pathologically modified Tau
Nature communications. 2022. Moll, A et al. Max Planck Inst Multidisciplinary Sci, Dept NMR Based Struct Biol, Fassberg 11, D-37077 Gottingen, Germany; German Ctr Neurodegenerat Dis DZNE, Von Siebold Str 3a, D-37075 Gottingen, Germany
ABSTRACT: Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region.
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Architecture of the human NALCN channelosome
CELL DISCOVERY. 2022. Zhou, LN et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou, Zhejiang, Peoples R China
ABSTRACT: NALCN regulates the resting membrane potential by mediating the Na+ leak current in neurons, and it functions as a channelosome in complex with FAM155A, UNC79, and UNC80. Dysfunction of the NALCN channelosome causes a broad range of neurological and developmental diseases called NALCN channelopathies in humans. How the auxiliary subunits, especially the two large components UNC79 and UNC80, assemble with NALCN and regulate its function remains unclear. Here we report an overall architecture of the human NALCN channelosome.
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Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies
Analytical Chemistry. 2022. Ruwolt, M et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful method for theinvestigation of protein-protein interactions (PPI) from highly complex samples. XL-MScombined with tandem mass tag (TMT) labeling holds the promise of large-scale PPIquantification. However, a robust and efficient TMT-based XL-MS quantificationmethod has not yet been established due to the lack of a benchmarking dataset andthorough evaluation of various MS parameters. To tackle these limitations, we generate atwo-interactome dataset by spiking in TMT-labeled cross-linkedEscherichia colilysateinto TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme.
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Structural basis for c-di-AMP–dependent regulation of the bacterial stringent response by receptor protein DarB
Journal of Biological Chemistry. 2022. Heidemann, JL et al. Georg August Univ Gottingen, Dept Mol Struct Biol, Gottingen, Germany
ABSTRACT: The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeo-stasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as poten-tial targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress condi-tions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP.
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Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL)
Journal of Extracellular Vesicles. 2022. Bauza-Martinez, J et al. ASTAR, Singapore Immunol Network SIgN, Singapore, Singapore
ABSTRACT: Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs.
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Real-time library search increases cross-link identification depth across all levels of sample complexity
Analytical chemistry. 2022. Ruwolt, Max et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany; Charite Univ Med Berlin, D-10117 Berlin, Germany
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition.
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Nitrogen Mustard Alkylates and Cross-Links p53 in Human Keratinocytes
Chemical Research in Toxicology. 2022. Jan, YH et al. Rutgers State Univ, Sch Publ Hlth, Dept Environm & Occupat Hlth & Justice, Piscataway, NJ 08854 USA
ABSTRACT: Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress.
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Enhanced protein--protein interaction network construction promoted by in vivo cross-linking with acid-cleavable click-chemistry enrichment
Frontiers in Chemistry. 2022. Zhao, Lili et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian, Liaoning, Peoples R China
ABSTRACT: Chemical cross-linking coupled with mass spectrometry has emerged as a powerful strategy which enables global profiling of protein interactome with direct interaction interfaces in complex biological systems. The alkyne-tagged enrichable cross-linkers are preferred to improve the coverage of low-abundance cross-linked peptides, combined with click chemistry for biotin conjugation to allow the cross-linked peptide enrichment. However, a systematic evaluation on the efficiency of click approaches (protein-based or peptide-based) and diverse cleavable click-chemistry ligands (acid, reduction, and photo) for cross-linked peptide enrichment and release is lacking.
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Structures of transcription preinitiation complex engaged with the+ 1 nucleosome
Nature Structural & Molecular Biology. 2022. Wang, HB et al. Max Planck Inst Multidisciplinary Sci, Dept Mol Biol, Gottingen, Germany
ABSTRACT: The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles within chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is located downstream of the core promoter. Here we present cryo-EM structures of the yeast PIC bound to promoter DNA and the +1 nucleosome located at three different positions.
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A toxin-deformation dependent inhibition mechanism in the T7SS toxin-antitoxin system of Gram-positive bacteria
Nature Communications. 2022. Wang, YJ et al. Jinan Univ, Coll Pharm, Int Cooperat Lab Tradit Chinese Med Modernizat &, Chinese Minist Educ MOE, Guangzhou 510632, Peoples R China; Weill Cornell Med, Dept Physiol & Biophys, New York, NY 10065 USA; Guangdong Youmei Inst Intelligent Biomfg, Foshan 528200, Guangdong, Peoples R China
ABSTRACT: Toxin EsaD secreted by some S. aureus strains through the type VII secretion system (T7SS) specifically kills those strains lacking the antitoxin EsaG. Here we report the structures of EsaG, the nuclease domain of EsaD and their complex, which together reveal an inhibition mechanism that relies on significant conformational change of the toxin. To inhibit EsaD, EsaG breaks the nuclease domain of EsaD protein into two independent fragments that, in turn, sandwich EsaG. The originally well-folded beta beta alpha-metal finger connecting the two fragments is stretched to become a disordered loop, leading to disruption of the catalytic site of EsaD and loss of nuclease activity.
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Mapping of the plant SnRK1 kinase signalling network reveals a key regulatory role for the class II T6P synthase-like proteins
Nature Plants. 2022. Van Leene, J et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium; VIB Ctr Plant Syst Biol, Ghent, Belgium
ABSTRACT: The central metabolic regulator SnRK1 controls plant growth and survival upon activation by energy depletion, but detailed molecular insight into its regulation and downstream targets is limited. Here we used phosphoproteomics to infer the sucrose-dependent processes targeted upon starvation by kinases as SnRK1, corroborating the relation of SnRK1 with metabolic enzymes and transcriptional regulators, while also pointing to SnRK1 control of intracellular trafficking. Next, we integrated affinity purification, proximity labelling and crosslinking mass spectrometry to map the protein interaction landscape, composition and structure of the SnRK1 heterotrimer, providing insight in its plant-specific regulation.
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Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry
Analytical Chemistry. 2022. Sun, MZ et al. Chinese Inst Brain Res CIBR, Beijing 102206, Peoples R China; Peking Univ, Synthet & Funct Biomol Ctr, Coll Chem & Mol Engn, Dept Chem Biol,Beijing Natl Lab Mol Sci,Key Lab Bi, Beijing 100871, Peoples R China; Inst Canc Res, Shenzhen Bay Lab, Shenzhen 518107, Peoples R China; Peking Univ, Peking Tsinghua Ctr Life Sci, Beijing 100871, Peoples R China; Peking Univ, PKU IDG McGovern Inst Brain Res, Beijing 100871, Peoples R China
ABSTRACT: Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation.
[more...]
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Genetically encoded chemical crosslinking of carbohydrate
Nature Chemistry. 2022. Li, SS et al. Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA; Univ Calif San Francisco, Cardiovasc Res Inst, Box 0544, San Francisco, CA 94143 USA
ABSTRACT: Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins.
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Divergent polo boxes in KKT2 bind KKT1 to initiate the kinetochore assembly cascade in Trypanosoma brucei
Molecular Biology of the Cell. 2022. Ishii, Midori et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England
ABSTRACT: Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases.
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Alkynyl-Enrichable Carboxyl-Selective Crosslinkers to Increase the Crosslinking Coverage for Deciphering Protein Structures
Analytical Chemistry. 2022. Gao, H et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: The coverage of chemical crosslinking coupled with mass spectrometry (CXMS) is of great importance to determine its ability for deciphering protein structures. At present, N- hydroxysuccinimidyl (NHS) ester-based crosslinkers targeting lysines have been predominantly used in CXMS. However, they are not always effective for some proteins with few lysines. Other amino acid residues such as carboxyl could be crosslinked to complement lysines and improve the crosslinking coverage of CXMS, but the low intrinsic chemical reactivity of carboxyl compromises the application of carboxyl-selective crosslinkers for complex samples.
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DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates
Iscience. 2022. Zhao, LJ et al. Chinese Acad Sci, Shanghai Inst Biochem & Cell Biol, Natl Ctr Prot Sci Shanghai, Ctr Excellence Mol Cell Sci, Shanghai 200031, Peoples R China; ShanghaiTech Univ, Sch Life Sci & Technol, 100 Haike Rd, Shanghai 201210, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China
ABSTRACT: Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyl-transferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two com-plementary mechanisms: A nucleosome-independent mechanism and a nucleo-some-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions.
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Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD
Nature. 2022. Su, SC et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Minist Educ,Key Lab Prot Sci, Beijing, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys, Collaborat Innovat Ctr Genet & Dev, Shanghai, Peoples R China
ABSTRACT: Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes(1,2). In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)(3,4). ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes(5,6). Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate.
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High-resolution mass spectrometry unveils the molecular changes of ovalbumin induced by heating and their influence on IgE binding capacity
Food Chemistry. 2022. Cherkaoui, M et al. INRAE, UR1268 BIA, F-44316 Nantes, France
ABSTRACT: Ovalbumin (OVA) is a food allergen whose allergenicity is modulated by heating. We aimed to establish a molecular connection between heat-induced structural modifications and the modulation of the IgE binding capacity of OVA. For this, we used model samples of heat-modified OVA with increasing complexity; glycated, aggregated, or glycated and aggregated. Using sera from egg-allergic individuals, we show that both aggregation and glycation strongly impacted IgE binding capacity, despite limited structural changes for glycated OVA.
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Pathway and mechanism of tubulin folding mediated by TRiC/CCT conjugated with its ATPase cycle revealed by cryo-EM
Communications Biology. 2022. Caixuan Liu et al. State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, 200031, Shanghai, China
ABSTRACT:
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Cryo-EM structures of human m6A writer complexes
Cell Research. 2022. Su, SC et al. Univ Sci & Technol China, MOE Key Lab Cellular Dynam, Hefei, Anhui, Peoples R China; Fudan Univ, Sch Life Sci, Dept Biochem & Biophys,Multiscale Res Inst Comple, State Key Lab Genet Engn,Collaborat Innovat Ctr G, Shanghai, Peoples R China; Univ Sci & Technol China, Div Life Sci & Med, Hefei, Anhui, Peoples R China
ABSTRACT: N-6-methyladenosine (m(6)A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m(6)A "writer" consists of the catalytic subunit m(6)A-METTL complex (MAC) and the regulatory subunit m(6)A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-angstrom resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA.
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Comprehensive structure and functional adaptations of the yeast nuclear pore complex
Cell. 2022. Akey, Christopher W. et al. Univ Calif San Diego, Div Biol Sci, Sect Mol Biol, La Jolla, CA 92093 USA; Univ Basque Country, Inst Biofis, UPV EHU, CSIC, Leioa 48940, Spain; Univ Calif San Diego, Howard Hughes Med Inst, La Jolla, CA 92093 USA; Baylor Coll Med, Verna & Marrs McLean Dept Biochem & Mol Biol, 1 Baylor Plaza, Houston, TX 77030 USA; Rockefeller Univ, Lab Cellular & Struct Biol, New York, NY 10065 USA; Basque Fdn Sci, Ikerbasque, Bilbao 48013, Spain
ABSTRACT: Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport.
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Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism
Nature structural & molecular biology. 2022. Chang, YG et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic, Australia
ABSTRACT: P-Rex (PI(3,4,5)P-3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by G beta gamma and PI(3,4,5)P-3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain.
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Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32
Science Advances. 2022. Mi, Chenchen et al. Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Guangdong, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Beijing Frontier Res Ctr Biol Struct, Sch Life Sci,State Key Lab Membrane Biol, Beijing 100084, Peoples R China
ABSTRACT: Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms-core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation.
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Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor
Science China Life Sciences. 2022. Huang, XX et al. Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, CAS Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China
ABSTRACT: Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor.
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Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability
PNAS. 2022. Hodson, Charlotte et al. Univ Melbourne, Dept Med St Vincents, Fitzroy, Vic 3065, Australia; St Vincents Inst Med Res, Genome Stabil Unit, Fitzroy, Vic 3065, Australia
ABSTRACT: The RecQ-like helicase BLM cooperates with topoisomerase IIIa, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIa are coupled, we purified the active four-subunit complex.
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Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology
Elife. 2022. Zhang, SN et al. Shanghai Jiao Tong Univ, Bio X Inst, Key Lab Genet Dev & Neuropsychiat Disorders, Minist Educ, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China; Univ Miami, Miller Sch Med, Dept Mol & Cellular Pharmacol, Miami, FL 33136 USA
ABSTRACT: Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer's disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD.
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SpotLink enables sensitive and precise identification of site nonspecific cross-links at the proteome scale
Briefings in Bioinformatics. 2022. Zhang, Weijie et al. Beihang Univ, Sch Biol Sci & Med Engn, Beijing 10010, Peoples R China; Beihang Univ, Sch Engn Med, Beijing 10010, Peoples R China; Chinese Acad Sci, Key Lab Separat Sci Analyt Chem, Dalian Inst Chem Phys, Dalian 116023, Peoples R China
ABSTRACT: Nonspecific cross-linker can provide distance restraints between surface residues of any type, which could be used to investigate protein structure construction and protein-protein interaction (PPI). However, the vast number of potential combinations of cross-linked residues or sites obtained with such a cross-linker makes the data challenging to analyze, especially for the proteome-wide applications. Here, we developed SpotLink software for identifying site nonspecific cross-links at the proteome scale.
[more...]
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Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis
Analytical Chemistry. 2022. An, YX et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Liaoning, Peoples R China
ABSTRACT: Chemical crosslinking combined with mass spectrometry (CXMS) has allowed the global characterization of protein complexes with high throughput and accuracy. Although enrichable crosslinkers have been introduced to exclude the interference of regular peptides, the crosslinked peptide identification is still severely inhibited by a large amount of monolinked peptides. In this work, we proposed a strategy called MoTE (unhydrolyzed Monolinked peptide Targeting Elimination) to remove the unhydrolyzed monolinked peptides, while enriching crosslinked peptides for regular peptide removal.
[more...]
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The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins
Journal of Biological Chemistry. 2022. Huang, CA et al. Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Renji Hosp Res Ctr, Renji Hosp, Sch Med, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Biox Inst, Minist Educ, Key Lab Genet Dev & Neuropsychiat Disorders, Shanghai, Peoples R China; Shanghai Jiao Tong Univ, Zhangjiang Inst Adv Study, Shanghai, Peoples R China
ABSTRACT: Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD(+) synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including alpha-synuclein, Tau (K19), amyloid beta, and islet amyloid polypeptide.
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D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply …
International Journal of Mass Spectrometry. 2022. Zuo, Mei-Qing et al. Natl Inst Biol Sci, Beijing, Peoples R China
ABSTRACT: Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not.
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Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition
Journal of Cell Biology. 2022. Jiang, QQ et al. Univ Penn, Penn Ctr Genome Integr, Basser Ctr BRCA, Perelman Sch Med,Dept Canc Biol, Philadelphia, PA 19104 USA; Univ Leeds, Fac Biol Sci, Astbury Ctr Struct Mol Biol, Sch Mol & Cellular Biol, Leeds, W Yorkshire, England
ABSTRACT: The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36.
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D/E-rich peptides are less suitable than D/E-deficient peptides for identification by negative-ion HCD due to scarce production of sequencing ions from multiply charged precursors
International Journal of Mass Spectrometry. 2022. Zuo, Mei-Qing et al. Natl Inst Biol Sci, Beijing, Peoples R China
ABSTRACT: Highly acidic, D/E-rich peptides or proteins are difficult to identify by positive-ion-mode mass spec-trometry (MS), and negative-ion-mode MS is an attractive but insufficiently explored alternative. Based on high-resolution and accurate-mass MS analysis of 115 synthetic peptides of 5-28 amino acids, we confirmed that higher-energy collisional dissociation (HCD) of deprotonated peptides induced abundant backbone or side-chain neutral losses (NL), and updated the ranking list of NLs by abundance. The most abundant fragment ion types are y-> x-, z-> c-if the NL ions are included, or c-> y-> z-> 6-if not.
[more...]
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Optimized TMT-Based Quantitative Cross-Linking Mass Spectrometry Strategy for Large-Scale Interactomic Studies
Analytical Chemistry. 2022. Ruwolt, M et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Struct Biol, D-13125 Berlin, Germany
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is a powerful method for theinvestigation of protein-protein interactions (PPI) from highly complex samples. XL-MScombined with tandem mass tag (TMT) labeling holds the promise of large-scale PPIquantification. However, a robust and efficient TMT-based XL-MS quantificationmethod has not yet been established due to the lack of a benchmarking dataset andthorough evaluation of various MS parameters. To tackle these limitations, we generate atwo-interactome dataset by spiking in TMT-labeled cross-linkedEscherichia colilysateinto TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme.
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Multiaspect examinations of possible alternative mappings of identified variant peptides: a case study on the HEK293 cell line
ACS omega. 2022. Choong, WK et al. Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan
ABSTRACT: Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking.
[more...]
Use: pNovo



Identification of Per a 13 as a novel allergen in American cockroach
Molecular immunology. 2022. Xu, ZQ et al. Nanjing Med Univ, Dept Pharm, Jiangsu Canc Hosp, Affiliated Canc Hosp,Jiangsu Inst Canc Res, 42 Baiziting Rd, Nanjing 210029, Peoples R China; Nanjing Med Univ, Childrens Hosp, Dept Resp Med, 72 Guangzhou Rd, Nanjing 210000, Peoples R China; Chinese Acad Med Sci & Peking Union Med Coll, Peking Union Med Coll Hosp, State Key Lab Complex Severe & Rare Dis, Dept Allergy, 1 Shuaifuyuan, Beijing 100730, Peoples R China
ABSTRACT: Background: Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach.Methods: The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning.
[more...]
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A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms
Molecular Immunology. 2022. Ling, XJ et al. Gannan Med Univ, Dept Blood Transfus, Affiliated Hosp 1, Ganzhou 341000, Jiangxi, Peoples R China; Nanjing Med Univ, Jiangsu Canc Hosp, Dept Pharm, Nanjing 210009, Peoples R China; Peking Union Med Coll Hosp, Chinese Acad Med Sci & Peking Union Med Coll, State Key Lab Complex Severe & Rare Dis, Allergy Dept, Beijing 100730, Peoples R China; Nanjing Med Univ, Affiliated Canc Hosp, Nanjing 210009, Peoples R China; Nanjing Med Univ, Jiangsu Inst Canc Res, Nanjing 210009, Peoples R China
ABSTRACT: Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteineprotease (CP) allergen from giant ragweed pollen, named as Amb t CP.
[more...]
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Multiaspect Examinations of Possible Alternative Mappings of Identified Variant Peptides: A Case Study on the HEK293 Cell Line
ACS omega. 2022. Choong, WK et al. Acad Sinica, Inst Informat Sci, Taipei 11529, Taiwan
ABSTRACT: Adopting proteogenomics approach to validatesingle nucleotide variation events by identifying correspondingsingle amino acid variant peptides from mass spectrometry (MS)-based proteomics data facilitates translational and clinical research.Although variant peptides are usually identified from MS data witha stringent false discovery rate (FDR), FDR control could fail toeliminate dubious results caused by several issues; thus,postexamination to eliminate dubious results is required. However,comprehensive postexaminations of identification results are stilllacking.
[more...]
Use: pNovo



Highly robust de novo full-length protein sequencing
Analytical Chemistry. 2022. Mai, NB et al. Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Peoples R China; Jinan Univ, MOE Key Lab Tumor Mol Biol, Inst Life & Hlth Engn, Guangzhou 510632, Peoples R China
ABSTRACT: Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods. De novo identified peptide sequence largely contain errors, undermining the accuracy of assembly. Bias on the detectability of the peptides also makes low-coverage regions, resulting in gaps. Although recent advances on multi-enzyme hydrolysis and algorithms showed complete assembly of full-length protein sequences in a few examples, the robustness in practical application is still to be improved.
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Use: pNovo; pParse



Highly Robust de Novo Full-Length Protein Sequencing
Analytical Chemistry. 2022. Mai, NB et al. Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Peoples R China; Jinan Univ, MOE Key Lab Tumor Mol Biol, Inst Life & Hlth Engn, Guangzhou 510632, Peoples R China
ABSTRACT: Accurate full-length sequencing of a purified unknown protein is still challenging nowadays due to the error-prone mass-spectrometry (MS)-based methods. De novo identified peptide sequence largely contain errors, undermining the accuracy of assembly. Bias on the detectability of the peptides also makes low-coverage regions, resulting in gaps. Although recent advances on multi-enzyme hydrolysis and algorithms showed complete assembly of full-length protein sequences in a few examples, the robustness in practical application is still to be improved.
[more...]
Use: pNovo; pParse



Diversity matters: optimal collision energies for tandem mass spectrometric analysis of a large set of N-glycopeptides
Journal of Proteome Research. 2022. Hever, H et al. Res Ctr Nat Sci, Eotvos Lorand Res Network, MS Prote Res Grp, H-1117 Budapest, Hungary
ABSTRACT: Identification and characterization of N-glycopep-tides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument.
[more...]
Use: pGlyco



Diversity Matters: Optimal Collision Energies for Tandem Mass Spectrometric Analysis of a Large Set of N-Glycopeptides
Journal of Proteome Research. 2022. Hever, H et al. Res Ctr Nat Sci, Eotvos Lorand Res Network, MS Prote Res Grp, H-1117 Budapest, Hungary
ABSTRACT: Identification and characterization of N-glycopep-tides from complex samples are usually based on tandem mass spectrometric measurements. Experimental settings, especially the collision energy selection method, fundamentally influence the obtained fragmentation pattern and hence the confidence of the database search results ("score"). Using standards of naturally occurring glycoproteins, we mapped the Byonic and pGlyco search engine scores of almost 200 individual N-glycopeptides as a function of collision energy settings on a quadrupole time of flight instrument.
[more...]
Use: pGlyco



Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism
Molecular Therapy-Nucleic Acids. 2022. Wang, YQ et al. East China Univ Sci & Technol, Sch Bioengn, Shanghai 200237, Peoples R China; East China Univ Sci & Technol, Sch Pharm, Shanghai 200237, Peoples R China
ABSTRACT: Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is rela-tively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we per formed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2.
[more...]
Use: pGlyco



TMT-based multiplexed quantitation of N-glycopeptides reveals glycoproteome remodeling induced by oncogenic mutations
ACS omega. 2022. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA; Inst Bioinformat, Bangalore 560066, Karnataka, India; Manipal Acad Higher Educ MAHE, Manipal 576104, Karnataka, India; Natl Inst Mental Hlth & Neurosci NIMHANS, Ctr Mol Med, Bangalore 560029, Karnataka, India; Mayo Clin, Ctr Individualized Med, Rochester, MN 55905 USA
ABSTRACT: Y Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome.
[more...]
Use: pGlyco



GlycAP, a glycoproteomic analysis platform for site-specific N-glycosylation research
International Journal of Mass Spectrometry. 2022. Wu, Mengxi et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China; Fudan Univ, Shanghai Peoples Hosp 5, Dept Chem, Shanghai, Peoples R China
ABSTRACT: Protein glycosylation is of great importance for its strong association with various diseases. Mass spectrometry-based site-specific glycoproteome methods with efficient interpretation software tools have become powerful strategies for glycosylation research. However, the lack of bioinformatics tools for automatic analysis of the interpretation data hinders further exploration. Here, we developed a comprehensive N-glycoproteomic analysis platform called GlycAP, which is embedded with different analytical modules, including qualitative analysis, quantitative analysis, functional analysis, and clinical analysis.
[more...]
Use: pGlyco



N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts
Journal of inherited metabolic disease. 2022. Budhraja, R et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA; Mayo Clin, Dept Lab Med & Pathol, 200 First St SW, Rochester, MN 55905 USA
ABSTRACT: Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated.
[more...]
Use: pGlyco



Multiattribute glycan identification and FDR control for glycoproteomics
Molecular & Cellular Proteomics. 2022. Polasky, Daniel A. et al. Univ Michigan, Dept Computat Med & Bioinformat, Ann Arbor, MI 48109 USA; Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA
ABSTRACT: Rapidly improving methods for glycoproteomics have enabled increasingly large-scale analyses of complex glycopeptide samples, but annotating the resulting mass spectrometry data with high confidence remains a major bottleneck. We recently introduced a fast and sensitive glycoproteomics search method in our MSFragger search engine, which reports glycopeptides as a combination of a peptide sequence and the mass of the attached glycan. In samples with complex glycosylation patterns, converting this mass to a specific glycan composition is not straightforward; however, as many glycans have similar or identical masses.
[more...]
Use: pGlyco



Exploration of quantitative site-specific serum O-glycoproteomics with isobaric labeling for the discovery of putative O-glycoprotein biomarkers
PROTEOMICS--Clinical Applications. 2022. Zhang, Zihan et al. Shanghai Jiao Tong Univ, Sch Med, Shanghai Peoples Hosp 9, Dept Plast & Reconstruct Surg, Shanghai 200011, Peoples R China; Tongji Univ, Shanghai Key Lab Chem Assessment & Sustainabil, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China
ABSTRACT: Purpose Exploration study of site-specific isobaric-TMT-labeling quantitative serum O-glycoproteomics for the discovery of putative O-glycoprotein cancer biomarkers. Experimental design Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N-glycosylation was first removed with PNGase F. After tryptic digestion of de-N-glycosylated serum proteome, the TMT-labeled O-glycopeptides mixture was prepared and analyzed with RPLC-MS/MS. Site-specific qualitative and quantitative database search of O-glycopeptides was carried out with pGlyco 3.0.
[more...]
Use: pGlyco



TMT-Based Multiplexed Quantitation of N-Glycopeptides Reveals Glycoproteome Remodeling Induced by Oncogenic Mutations
ACS omega. 2022. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA; Inst Bioinformat, Bangalore 560066, Karnataka, India; Manipal Acad Higher Educ MAHE, Manipal 576104, Karnataka, India; Natl Inst Mental Hlth & Neurosci NIMHANS, Ctr Mol Med, Bangalore 560029, Karnataka, India; Mayo Clin, Ctr Individualized Med, Rochester, MN 55905 USA
ABSTRACT: Y Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome.
[more...]
Use: pGlyco



Structure-Specific N-Glycoproteomics Characterization of NIST Monoclonal Antibody Reference Material 8671
Journal of Proteome Research. 2022. Bi, M et al. Tongji Univ, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China; Nanjing Univ, Ctr Precis Med, Dept Lab Med, Affiliated Hosp,Med Sch,Nanjing Drum Tower Hosp, Nanjing 210008, Jiangsu, Peoples R China
ABSTRACT: The characteristics of monoclonal antibodies (mAbs) cohering various function effectors show great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There has been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentation of N-glycan moieties.
[more...]
Use: pGlyco



A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis
Molecular metabolism. 2022. Verzijl, CRC et al. Univ Med Ctr Groningen, Dept Pediat, Sect Mol Genet, Antonius Deusinglaan 1, NL-9713 AV Groningen, Netherlands
ABSTRACT: Objective: GALNT2, encoding polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2), was initially discovered as a regulator of highdensity lipoprotein metabolism. GalNAc-T2 is known to exert these effects through post-translational modification, i.e., O-linked glycosylation of secreted proteins with established roles in plasma lipid metabolism. It has recently become clear that loss of GALNT2 in rodents, cattle, nonhuman primates, and humans should be regarded as a novel congenital disorder of glycosylation that affects development and body weight.
[more...]
Use: pGlyco



Nascent glycoproteome reveals that N-linked glycosylation inhibitor-1 suppresses expression of glycosylated lysosome-associated membrane protein-2
Frontiers in Molecular Biosciences. 2022. Cao, Xinyi et al. Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China
ABSTRACT: Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line.
[more...]
Use: pQuant



Reaction-based fluorogenic probes for detecting protein cysteine oxidation in living cells
Nature Communications. 2022. Ferreira, RB et al. UF Scripps Biomed Res, Dept Chem, Jupiter, FL 33458 USA
ABSTRACT: Fluorogenic detection of H2O2 in cells is established, but equivalent tools to monitor its cellular targets remain in their infancy. Here authors develop fluorogenic probes for detecting cysteine sulfenic acid, a redox modification inextricably linked to H2O2 signalling and oxidative stress.'Turn-on' fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress.
[more...]
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Identification of NLRP3 as a covalent target of 1, 6-O, O-diacetylbritannilactone against neuroinflammation by quantitative thiol reactivity profiling (QTRP)
Bioorganic Chemistry. 2022. Wang, MR et al. Northwest A&F Univ, Coll Chem & Pharm, Shaanxi Key Lab Nat Prod & Chem Biol, 3 Taicheng Rd, Yangling 712100, Shaanxi, Peoples R China; Hainan Univ, Sch Pharmaceut Sci, Key Lab Trop Biol Resources, Minist Educ, Haikou 570228, Hainan, Peoples R China
ABSTRACT: Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative dis-eases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula bri-tannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/ NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear.
[more...]
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Nascent glycoproteome reveals that N-linked Glycosylation Inhibitor-1 Suppresses Expression of Glycosylated Lysosome-Associated Membrane Protein-2
Frontiers in Molecular Biosciences. 2022. Cao, Xinyi et al. Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China
ABSTRACT: Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line.
[more...]
Use: pQuant



Comparing top-down proteoform identification: Deconvolution, PrSM overlap, and PTM detection
. 2022. David L. Tabb et al. Université Paris Cité, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop



Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection
. 2022. David L. Tabb et al. Université Paris Cité, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop




2021




Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis
ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2021. Gonz{\'a}lez, Luis Javier et al. Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), Avenida 31, e/ 158 y 190, Cubanacán, Playa, 10600 Havana, Cuba
ABSTRACT: A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys(1)pP0 and p64K-beta Ala(1)pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0.
[more...]
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caAtlas: an immunopeptidome atlas of human cancer
Iscience. 2021. Yi, XP et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: Comprehensive characterization of tumor antigens is essential for the design of cancer immunotherapies, and mass spectrometry (MS)-based immunopeptidomics enables high-throughput identification of major histocompatibility complex (MHC)-bound peptide antigens in vivo. Here we construct an immunopeptidome atlas of human cancer through an extensive collection of 43 published immunopeptidomic datasets and standardized analysis of 81.6 million MS/MS spectra using an open search engine. Our analysis greatly expands the current knowledge of MHC-bound antigens, including an unprecedented characterization of post-translationally modified antigens and their cancer-association.
[more...]
Use: pFind



ADAP Y571 phosphorylation is required to prime STAT3 for activation in TLR4-stimulated macrophages
JOURNAL OF IMMUNOLOGY. 2021. Yang, Naiqi et al. Soochow Univ, Inst Biol, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China; Soochow Univ, Inst Med Sci, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China
ABSTRACT: Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS.
[more...]
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Acyl carrier protein promotes MukBEF action in Escherichia coli chromosome organization-segregation
Nature Communications. 2021. Prince, JP et al. Univ Oxford, Dept Biochem, South Parks Rd, Oxford OX1 3QU, England.
ABSTRACT: Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE.
[more...]
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Proteogenomic analysis provides novel insight into genome annotation and nitrogen metabolism in Nostoc Sp. PCC 7120
Microbiology Spectrum. 2021. Yu, Shengchao et al. Univ Chinese Acad Sci, Beijing, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China
ABSTRACT: Cyanobacteria, capable of oxygenic photosynthesis, play a vital role in nitrogen and carbon cycles. Nostoc sp. PCC 7120 (Nostoc 7120) is a model cyanobacterium commonly used to study cell differentiation and nitrogen metabolism. Although its genome was released in 2002, a high-quality genome annotation remains unavailable for this model cyanobacterium. Therefore, in this study, we performed an in-depth proteogenomic analysis based on high-resolution mass spectrometry (MS) data to refine the genome annotation of Nostoc 7120.
[more...]
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ADAP Y571 Phosphorylation Is Required to Prime STAT3 for Activation in TLR4-Stimulated Macrophages
JOURNAL OF IMMUNOLOGY. 2021. Yang, Naiqi et al. Soochow Univ, Inst Biol, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China; Soochow Univ, Inst Med Sci, 199 Renai Rd, Suzhou 215123, Jiangsu, Peoples R China
ABSTRACT: Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS.
[more...]
Use: pFind



Ribosome-amplified metabolism, RAMBO, measured by NMR spectroscopy
Biochemistry. 2021. Yu, JianChao et al. SUNY Albany, Dept Chem, Albany, NY 12222 USA
ABSTRACT: NMR spectroscopy was used to investigate the phenomenon of ribosome-amplified metabolism or RAMBO between pyruvate kinase and ribosomes. Because the concentration of ribosomes increases as the cell grows, ribosome binding interactions may regulate metabolic fluxes by altering the distribution of bound and free enzymes. Pyruvate kinase (PK) catalyzes the last step of glycolysis and represents a major drug target for controlling bacterial infections. The binding of metabolic enzymes to ribosomes creates protein quinary structures with altered catalytic activities.
[more...]
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Insulin signaling regulates longevity through protein phosphorylation in Caenorhabditis elegans
Nature Communications. 2021. Li, WJ et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Insulin/IGF-1 Signaling (IIS) is known to constrain longevity by inhibiting the transcription factor FOXO. How phosphorylation mediated by IIS kinases regulates lifespan beyond FOXO remains unclear. Here, we profile IIS-dependent phosphorylation changes in a large-scale quantitative phosphoproteomic analysis of wild-type and three IIS mutant Caenorhabditis elegans strains. We quantify more than 15,000 phosphosites and find that 476 of these are differentially phosphorylated in the long-lived daf-2/insulin receptor mutant.
[more...]
Use: pQuant; pFind



Structure of the activated human minor spliceosome
Science. 2021. Bai, Rui et al. Westlake Lab Life Sci & Biomed, Hangzhou 310024, Zhejiang, Peoples R China; Tsinghua Univ, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing Frontier Res Ctr Biol Struct, Beijing 100084, Peoples R China; Tsinghua Univ, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing Adv Innovat Ctr Struct Biol, Beijing 100084, Peoples R China; Westlake Inst Adv Study, Inst Biol, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China
ABSTRACT: The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the g-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP.
[more...]
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Proteogenomic characterization of the pathogenic fungus Aspergillus flavus reveals novel genes involved in aflatoxin production
Molecular & Cellular Proteomics. 2021. Yang, Mingkun et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Peoples R China; Fujian Agr & Forestry Univ, Key Lab Pathogen Fungi & Mycotoxins Fujian Prov, Fuzhou, Peoples R China; Fujian Agr & Forestry Univ, Sch Life Sci, Fuzhou, Peoples R China
ABSTRACT: Aspergillus flavus (A. flavus), a pathogenic fungus, can produce carcinogenic and toxic aflatoxins that are a serious agricultural and medical threat worldwide. Attempts to decipher the aflatoxin biosynthetic pathway have been hampered by the lack of a high-quality genome annotation for A. flavus. To address this gap, we performed a comprehensive proteogenomic analysis using high-accuracy mass spectrometry data for this pathogen. The resulting high-quality data set confirmed the translation of 8724 previously predicted genes and identified 732 novel proteins, 269 splice variants, 447 single amino acid variants, 188 revised genes.
[more...]
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Antibody-free enrichment method for proteome-wide analysis of endogenous SUMOylation sites
Analytica Chimica Acta. 2021. Li, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: SUMOylation is a reversible post-translational modification that plays crucial roles in numerous cellular processes. Although anti-SUMO antibodies have been applied to analyze exogenous and endogenous SUMOylation, such immunoprecipitation enrichment strategy is applicable only for the enrichment of one specific SUMO type in mammalian cells, unable to map the global landscape of all endogenous SUMOylation simultaneously. To address this issue, we proposed an antibody-free strategy to enrich and profile endogenous SUMO1/2/3-modified peptides simultaneously.
[more...]
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HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells
Science. 2021. Yu, HY et al. Univ Calif San Diego, Ludwig Inst Canc Res, La Jolla, CA 92093 USA.
ABSTRACT: The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores.
[more...]
Use: pFind; pParse; pQuant



DeepLC can predict retention times for peptides that carry as-yet unseen modifications
Nature methods. 2021. Bouwmeester, R et al. VIB, VIB UGent Ctr Med Biotechnol, Ghent, Belgium.
ABSTRACT: DeepLC, a deep learning-based peptide retention time predictor, can predict retention times for unmodified peptides as well as peptides with previously unseen modifications. The inclusion of peptide retention time prediction promises to remove peptide identification ambiguity in complex liquid chromatography-mass spectrometry identification workflows. However, due to the way peptides are encoded in current prediction models, accurate retention times cannot be predicted for modified peptides. This is especially problematic for fledgling open searches, which will benefit from accurate retention time prediction for modified peptides to reduce identification ambiguity.
[more...]
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Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans
Nature communications. 2021. Meng, J et al. Joslin Diabet Ctr, Div Res, 1 Joslin Pl, Boston, MA 02215 USA.
ABSTRACT: Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment.
[more...]
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Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3. 4
Molecular cell. 2021. Green, MN et al. Columbia Univ, Dept Biochem & Mol Biophys, 650 West 168th St, New York, NY 10032 USA.
ABSTRACT: Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs).
[more...]
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Spatiotemporally resolved subcellular phosphoproteomics
PNAS. 2021. Liu, YJ et al. Peking Univ, PKU IDG McGovern Inst Brain Res, AH-100871 Beijing, Peoples R China.
ABSTRACT: Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial-temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers.
[more...]
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Wittig reagents for chemoselective sulfenic acid ligation enables global site stoichiometry analysis and redox-controlled mitochondrial targeting
Nature Chemistry. 2021. Shi, YL et al. Scripps Res Inst, Dept Chem, Jupiter, FL 33458 USA.
ABSTRACT: Triphenylphosphonium ylides, known as Wittig reagents, are one of the most commonly used tools in synthetic chemistry. Despite their considerable versatility, Wittig reagents have not yet been explored for their utility in biological applications. Here we introduce a chemoselective ligation reaction that harnesses the reactivity of Wittig reagents and the unique chemical properties of sulfenic acid, a pivotal post-translational cysteine modification in redox biology. The reaction, which generates a covalent bond between the ylide nucleophilic alpha-carbon and electrophilic gamma-sulfur, is highly selective, rapid and affords robust labelling under a range of biocompatible reaction conditions, which includes in living cells.
[more...]
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Potential use of serum proteomics for monitoring COVID-19 progression to complement RT-PCR detection
Journal of proteome research. 2021. Zhang, Y et al. Wenzhou Med Univ, Taizhou Hosp, Linhai 317000, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310000, Zhejiang, Peoples R China
ABSTRACT: RT-PCR is the primary method to diagnose COVID-19 and is also used to monitor the disease course. This approach, however, suffers from false negatives due to RNA instability and poses a high risk to medical practitioners. Here, we investigated the potential of using serum proteomics to predict viral nucleic acid positivity during COVID19. We analyzed the proteome of 275 inactivated serum samples from 54 out of 144 COVID-19 patients and shortlisted 42 regulated proteins in the severe group and 12 in the non-severe group.
[more...]
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Highly Efficient Enrichment of O-GlcNAc Glycopeptides Based on Chemical Oxidation and Reversible Hydrazide Chemistry
Analytical Chemistry. 2021. Chen, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China
ABSTRACT: Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups.
[more...]
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Dynamically remodeled hepatic extracellular matrix predicts prognosis of early-stage cirrhosis
Cell Death & Disease. 2021. Wu, YX et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Cell Biol, Shanghai 200031, Peoples R China.
ABSTRACT: Liver cirrhosis remains major health problem. Despite the progress in diagnosis of asymptomatic early-stage cirrhosis, prognostic biomarkers are needed to identify cirrhotic patients at high risk developing advanced stage disease. Liver cirrhosis is the result of deregulated wound healing and is featured by aberrant extracellular matrix (ECM) remodeling. However, it is not comprehensively understood how ECM is dynamically remodeled in the progressive development of liver cirrhosis. It is yet unknown whether ECM signature is of predictive value in determining prognosis of early-stage liver cirrhosis.
[more...]
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Determinants of genome-wide distribution and evolution of uORFs in eukaryotes
Nature communications. 2021. Zhang, H et al. Peking Univ, Sch Life Sci, Ctr Bioinformat, State Key Lab Prot & Plant Gene Res, Beijing, Peoples R China.
ABSTRACT: Upstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze similar to 17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (N-e), has shaped the contents of uORFs in eukaryotes.
[more...]
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Allosteric activation of SARS-CoV-2 RNA-dependent RNA polymerase by remdesivir triphosphate and other phosphorylated nucleotides
MBio. 2021. Wang, B et al. Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA.
ABSTRACT: The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity.
[more...]
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Expression profiles of antimicrobial peptides in Mytilus coruscus
Aquaculture. 2021. Yang, JY et al. Zhejiang Ocean Univ, Marine Sci & Tech Coll, Lab Marine Biol Prot Engn, Zhoushan City 316022, Zhejiang, Peoples R China
ABSTRACT: Antimicrobial peptides (AMPs) play a fundamental role in mussels' innate immunity, preventing the invasion of potential pathogens. Previous research has shown that AMPs are abundant in Mytilus species. A mussel with important economic value and limited distribution in the East China Sea, M. coruscus, also contains abundant AMPs, including the mytichitin and myticusin identified previously in this species. However, the molecular diversity and expression pattern of M. coruscus AMPs remain largely unknown.
[more...]
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ZmMPK5 phosphorylates ZmNAC49 to enhance oxidative stress tolerance in maize
New Phytologist. 2021. Xiang, Y et al. Nanjing Agr Univ, Coll Life Sci, Nanjing 210095, Jiangsu, Peoples R China.
ABSTRACT: Mitogen-activated protein kinase (MPK) is a critical regulator of the antioxidant defence system in response to various stimuli. However, how MPK directly and exactly regulates antioxidant enzyme activities is still unclear. Here, we demonstrated that a NAC transcription factor ZmNAC49 mediated the regulation of antioxidant enzyme activities by ZmMPK5. ZmNAC49 expression is induced by oxidative stress. ZmNAC49 enhances oxidative stress tolerance in maize, and it also reduces superoxide anion generation and increases superoxide dismutase (SOD) activity.
[more...]
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A20/Nrdp1 interaction alters the inflammatory signaling profile by mediating K48-and K63-linked polyubiquitination of effectors MyD88 and TBK1
Journal of Biological chemistry. 2021. Meng, ZY et al. Army Med Univ, Xinqiao Hosp, Dept Neurol, Mil Med Univ 3, Chongqing, Peoples R China.
ABSTRACT: A20 is a potent anti-inflammatory protein that mediates both inflammation and ubiquitination in mammals, but the related mechanisms are not clear. In this study, we performed mass spectrometry (MS) screening, gene ontology (GO) analysis, and coimmunoprecipitation (co-IP) in a lipopolysaccharide (LPS)-induced inflammatory cell model to identify novel A20-interacting proteins. We confirmed that the E3 ubiquitin ligase Nrdp1, also known as ring finger protein 41 (RNF41), interacted with A20 in LPS-stimulated cells.
[more...]
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caAtlas: An immunopeptidome atlas of human cancer
Iscience. 2021. Yi, XP et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: Comprehensive characterization of tumor antigens is essential for the design of cancer immunotherapies, and mass spectrometry (MS)-based immunopeptidomics enables high-throughput identification of major histocompatibility complex (MHC)-bound peptide antigens in vivo. Here we construct an immunopeptidome atlas of human cancer through an extensive collection of 43 published immunopeptidomic datasets and standardized analysis of 81.6 million MS/MS spectra using an open search engine. Our analysis greatly expands the current knowledge of MHC-bound antigens, including an unprecedented characterization of post-translationally modified antigens and their cancer-association.
[more...]
Use: pFind



Site-specific N-and O-glycosylation analysis of human plasma fibronectin
frontiers in Chemistry. 2021. Liu, D et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the O-18-labeling method.
[more...]
Use: pFind; pGlyco



A semi-tryptic peptide centric metaproteomic mining approach and its potential utility in capturing signatures of gut microbial proteolysis
MICROBIOME. 2021. Yan, ZX et al. Sun Yat Sen Univ, Affiliated Hosp 5, Guangdong Prov Key Lab Biomed Imaging, Zhuhai 519000, Guangdong, Peoples R China.
ABSTRACT: BackgroundProteolysis regulation allows gut microbes to respond rapidly to dynamic intestinal environments by fast degradation of misfolded proteins and activation of regulatory proteins. However, alterations of gut microbial proteolytic signatures under complex disease status such as inflammatory bowel disease (IBD, including Crohn's disease (CD) and ulcerative colitis (UC)), have not been investigated. Metaproteomics holds the potential to investigate gut microbial proteolysis because semi-tryptic peptides mainly derive from endogenous proteolysis.ResultsWe have developed a semi-tryptic peptide centric metaproteomic mining approach to obtain a snapshot of human gut microbial proteolysis signatures.
[more...]
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Exploring the microbiome-wide lysine acetylation, succinylation, and propionylation in human gut microbiota
Analytical Chemistry. 2021. Zhang, X et al. Univ Ottawa, Fac Med, Ottawa Inst Syst Biol, Ottawa, ON K1H 8M5, Canada.
ABSTRACT: Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples.
[more...]
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Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007—A Dominant Yeast in the Fermentation Process of Pu-erh Tea
Journal of proteome research. 2021. Tian, F et al. Yunnan Univ, Sch Life Sci, Yunnan Inst Microbiol, Minist Educ,Key Lab Microbial Divers Southwest Ch, Kunming 650091, Yunnan, Peoples R China.
ABSTRACT: Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES.
[more...]
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Mirror-Cutting-Based Digestion Strategy Enables the In-Depth and Accuracy Characterization of N-Linked Protein Glycosylation
Journal of proteome research. 2021. Chen, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: N-linked glycosylation plays important roles in multiple physiological and pathological processes, while the analysis coverage is still limited due to the insufficient digestion of glycoproteins, as well as incomplete ion fragments for intact glycopeptide determination. Herein, a mirror-cutting-based digestion strategy was proposed by combining two orthogonal proteases of LysargiNase and trypsin to characterize the macro- and micro-heterogeneity of protein glycosylation. Using the above two proteases, the b- or y-ion series of peptide sequences were, respectively, enhanced in MS/MS, generating the complementary spectra for peptide sequence identification.
[more...]
Use: pFind; pGlyco



DIA-based proteomics identifies IDH2 as a targetable regulator of acquired drug resistance in chronic myeloid leukemia
Molecular & Cellular Proteomics. 2021. Liu, W et al. Dalian Med Univ, Coll Pharm, Dept Clin Pharmacol, Dalian, Liaoning, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Zhejiang, Peoples R China
ABSTRACT: Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs defined by their increasing resistance index. PulseDIAbased (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells.
[more...]
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Proteogenomics Integrating Novel Junction Peptide Identification Strategy Discovers Three Novel Protein Isoforms of Human NHSL1 and EEF1B2
Journal of proteome research. 2021. He, Cuitong et al. Peking-Tsinghua Centre for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, 100871 Beijing, China
ABSTRACT: In eukaryotes, alternative pre-mRNA splicing allows a single gene to encode different protein isoforms that function in many biological processes, and they are used as biomarkers or therapeutic targets for diseases. Although protein isoforms in the human genome are well annotated, we speculate that some low-abundance protein isoforms may still be under-annotated because most genes have a primary coding product and alternative protein isoforms tend to be under-expressed. A peptide coencoded by a novel exon and an annotated exon separated by an intron is known as a novel junction peptide.
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An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry
JOURNAL OF PROTEOME RESEARCH. 2021. Guan, YD et al. Univ Med Ctr Hamburg Eppendorf, Inst Clin Chem & Lab Med, Sect Mass Spectrometr Prote, D-20246 Hamburg, Germany.
ABSTRACT: The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation).
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Potential Use of Serum Proteomics for Monitoring COVID-19 Progression to Complement RT-PCR Detection
Journal of proteome research. 2021. Zhang, Y et al. Wenzhou Med Univ, Taizhou Hosp, Linhai 317000, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou 310000, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310000, Zhejiang, Peoples R China
ABSTRACT: RT-PCR is the primary method to diagnose COVID-19 and is also used to monitor the disease course. This approach, however, suffers from false negatives due to RNA instability and poses a high risk to medical practitioners. Here, we investigated the potential of using serum proteomics to predict viral nucleic acid positivity during COVID19. We analyzed the proteome of 275 inactivated serum samples from 54 out of 144 COVID-19 patients and shortlisted 42 regulated proteins in the severe group and 12 in the non-severe group.
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Exploring the Microbiome-Wide Lysine Acetylation, Succinylation, and Propionylation in Human Gut Microbiota
Analytical Chemistry. 2021. Zhang, X et al. Univ Ottawa, Fac Med, Ottawa Inst Syst Biol, Ottawa, ON K1H 8M5, Canada.
ABSTRACT: Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples.
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Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry
Analytical chemistry. 2021. Riffle, M et al. Univ Washington, Dept Biochem, Seattle, WA 98195 USA
ABSTRACT: Drugs are often metabolized to reactive intermedi-ates that form protein adducts. Adducts can inhibit protein activity,elicit immune responses, and cause life-threatening adverse drugreactions. The masses of reactive metabolites are frequentlyunknown, rendering traditional mass spectrometry-based proteo-mics approaches incapable of adduct identification. Here, wepresent Magnum, an open-mass search algorithm optimized foradduct identification, and Limelight, a web-based data processingpackage for analysis and visualization of data from all existingalgorithms.
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DIA-based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia
Molecular & Cellular Proteomics. 2021. Liu, W et al. Dalian Med Univ, Coll Pharm, Dept Clin Pharmacol, Dalian, Liaoning, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou, Zhejiang, Peoples R China; Westlake Lab Life Sci & Biomed, Ctr Infect Dis Res, Hangzhou, Zhejiang, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou, Zhejiang, Peoples R China
ABSTRACT: Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs defined by their increasing resistance index. PulseDIAbased (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells.
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Site-Specific N-and O-Glycosylation Analysis of Human Plasma Fibronectin
frontiers in Chemistry. 2021. Liu, D et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the O-18-labeling method.
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Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by Remdesivir Triphosphate and Other Phosphorylated Nucleotides
MBio. 2021. Wang, B et al. Ohio State Univ, Dept Microbiol, Columbus, OH 43210 USA.
ABSTRACT: The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity.
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FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer
Nature communications. 2021. Huang, Y et al. Sun Yat Sen Univ Canc Ctr, Collaborat Innovat Ctr Canc Med, State Key Lab Oncol South China, Guangdong Key Lab Nasopharyngeal Carcinoma Diag &, Guangzhou, Peoples R China.
ABSTRACT: Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors.
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Leep1 interacts with PIP3 and the Scar/WAVE complex to regulate cell migration and macropinocytosis
JOURNAL OF CELL BIOLOGY. 2021. Yang, YH et al. Chinese Acad Sci, Ctr Excellence Biomacromol, Inst Biophys, Natl Lab Biomacromol, Beijing, Peoples R China.
ABSTRACT: Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator.
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Carboxypeptidase Y assisted disulfide-bond identification with linearized database search
Analytical Chemistry. 2021. Qiang, Jiali et al. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 100 Haike Road, Pudong, Shanghai 201210, China
ABSTRACT: A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)".
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Structures of the human Mediator and Mediator-bound preinitiation complex
Science. 2021. Chen, XZ et al. Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, State Key Lab Genet Engn,Shanghai Key Lab Radiat, Shanghai, Peoples R China.
ABSTRACT: The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PICMediator (76 polypeptides, similar to 4.1 megadaltons) in four distinct conformations.
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Covalently engineered nanobody chimeras for targeted membrane protein degradation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2021. Zhang, H et al. Peking Univ, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Beijing 100871, Peoples R China.
ABSTRACT: The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells.
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Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape
eLife. 2021. Mast, FD et al. Seattle Childrens Res Inst, Ctr Global Infect Dis Res, Seattle, WA 98101 USA; Rockefeller Univ, Lab Cellular & Struct Biol, 1230 York Ave, New York, NY 10021 USA; Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, 1230 York Ave, New York, NY 10021 USA; Univ Washington, Dept Pediat, Seattle, WA 98195 USA; Univ Washington, Dept Biochem, Seattle, WA 98195 USA
ABSTRACT: The emergence of SARS-CoV-2 variants threatens current vaccines and therapeutic antibodies and urgently demands powerful new therapeutics that can resist viral escape. We therefore generated a large nanobody repertoire to saturate the distinct and highly conserved available epitope space of SARS-CoV-2 spike, including the S1 receptor binding domain, N-terminal domain, and the S2 subunit, to identify new nanobody binding sites that may reflect novel mechanisms of viral neutralization. Structural mapping and functional assays show that indeed these highly stable monovalent nanobodies potently inhibit SARS-CoV-2 infection, display numerous neutralization mechanisms, are effective against emerging variants of concern, and are resistant to mutational escape.
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The structure of a virus-encoded nucleosome
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2021. Valencia-Sanchez, MI et al. New York Univ Grossman Sch Med, Skirball Inst Biomol Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT: The cryo-EM structure of DNA-assembled histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus, a nucleocytoplasmic large DNA virus, reveals that these proteins form viral nucleosomes with highly conserved features when compared to canonical eukaryotic nucleosomes. Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs H beta-H alpha and H delta-H gamma from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3.
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Chemical synthesis of activity‐based E2‐ubiquitin probes for the structural analysis of E3 ligase‐catalyzed transthiolation
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 2021. Liang, LJ et al. Tsinghua Univ, Tsinghua Peking Ctr Life Sci, Minist Educ,Dept Chem, Key Lab Bioorgan Phosphorus Chem & Chem Biol,Ctr, Beijing 100084, Peoples R China.
ABSTRACT: Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s.
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Diethynyl Phosphinates for Cysteine‐Selective Protein Labeling and Disulfide Rebridging
Angewandte Chemie International Edition. 2021. Stieger, CE et al. Forsch Verbund Berlin EV FMP, Leibniz Forschungsinst Mol Pharmakol, Chem Biol Dept, Campus Berlin Buch,Robert Roessle Str 10, D-13125 Berlin, Germany.
ABSTRACT: Diethynyl phosphinates were developed as bisfunctional electrophiles for the site-selective modification of peptides, proteins and antibodies. One of their electron-deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra- or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery.
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Molecular architecture of the endocytic TPLATE complex
Science Advances. 2021. Yperman, K et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Technol Pk 71, B-9052 Ghent, Belgium.
ABSTRACT: Eukaryotic cells rely on endocytosis to regulate their plasma membrane proteome and lipidome. Most eukaryotic groups, except fungi and animals, have retained the evolutionary ancient TSET complex as an endocytic regulator. Unlike other coatomer complexes, structural insight into TSET is lacking. Here, we reveal the molecular architecture of plant TSET [TPLATE complex (TPC)] using an integrative structural approach. We identify crucial roles for specific TSET subunits in complex assembly and membrane interaction.
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Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain
PNAS. 2021. Hevler, Johannes F. et al. Univ Utrecht, Utrecht Inst Pharmaceut Sci, NL-3584 CH Utrecht, Netherlands; Univ Utrecht, Bijvoet Ctr Biomol Res, Netherlands Prote Ctr, NL-3584 CH Utrecht, Netherlands; Univ Cologne, Cologne Excellence Cluster Cellular Stress Respon, D-50931 Cologne, Germany; Radboud Univ Nijmegen, Med Ctr, Radboud Inst Mol Life Sci, NL-6525 GA Nijmegen, Netherlands; Univ Utrecht, Utrecht Inst Pharmaceut Sci, Bijvoet Ctr Biomol Res, Biomol Mass Spectrometry & Prote, NL-3584 CH Utrecht, Netherlands
ABSTRACT: Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with similar to 10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM1(2) complex (PDBDEV_00000092).
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Structural basis of soluble membrane attack complex packaging for clearance
Nature Communications. 2021. Menny, A et al. Imperial Coll London, Dept Life Sci, Sir Ernst Chain Bldg, London SW7 2AZ, England.
ABSTRACT: To prevent unregulated complement activation, extracellular chaperones capture soluble precursors to the membrane attack complex (sMAC). Here, structural analysis of sMAC reveals how clusterin recognizes heterogeneous sMAC complexes and inhibits polymerization of complement protein C9. Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC).
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Retention time prediction using neural networks increases identifications in crosslinking mass spectrometry
Nature communications. 2021. Giese, SH et al. Tech Univ Berlin, Inst Biotechnol, Bioanalyt, Berlin, Germany.
ABSTRACT: Crosslinking mass spectrometry has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the mass spectra of crosslinked peptides limits the numbers of protein-protein interactions that can be confidently identified. Here, we leverage chromatographic retention time information to aid the identification of crosslinked peptides from mass spectra. Our Siamese machine learning model xiRT achieves highly accurate retention time predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E.
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MS Annika: A new cross-linking search engine
Journal of Proteome Research. 2021. Pirklbauer, GJ et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct.
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Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida
Open Biology. 2021. Tromer, EC et al. Univ Cambridge, Dept Biochem, Cambridge, England.
ABSTRACT: Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18.
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Allosteric transcription stimulation by RNA polymerase II super elongation complex
Molecular Cell. 2021. Chen, Y et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 A resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation.
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Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies
Cell Systems. 2021. Xiang, YF et al. Univ Pittsburgh, Dept Cell Biol, Pittsburgh, PA 15260 USA.
ABSTRACT: The antibody immune response is essential for the survival of mammals. However, we still lack a systematic understanding of the antibody repertoire. Here, we developed a proteomic strategy to survey, at an unprecedented scale, the landscape of antigen-engaged, circulating camelid heavy-chain antibodies, whose minimal binding fragments are called VHH antibodies or nanobodies. The sensitivity and robustness of this approach were validated with three antigens spanning orders of magnitude in immune responses; thousands of distinct, high-affinity nanobody families were reliably identified and quantified.
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Large-scale ratcheting in a bacterial DEAH/RHA-type RNA helicase that modulates antibiotics susceptibility
PNAS. 2021. Grass, LM et al. Free Univ Berlin, Inst Chem & Biochem, Lab Struct Biochem, D-14195 Berlin, Germany.
ABSTRACT: Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5 ' RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity.
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A resource of high-quality and versatile nanobodies for drug delivery
Iscience. 2021. Shen, ZL et al. Univ Pittsburgh, Dept Cell Biol, Pittsburgh, PA 15260 USA.
ABSTRACT: Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (Nb-HSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of Nb(HSA)s, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected Nb(HSA)s in an HSA mouse model by quantitative proteomics.
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Reverse chemical ecology suggests putative primate pheromones
Molecular Biology and Evolution. 2021. Zaremska, V et al. Austrian Inst Technol GmbH, Biosensor Technol, Tulln, Austria
ABSTRACT: Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned.
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Cryo-EM structure of the yeast TREX complex and coordination with the SR-like protein Gbp2
eLife. 2021. Xie, YH et al. Vanderbilt Univ, Dept Biochem, Sch Med, Nashville, TN 37232 USA.
ABSTRACT: The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Text, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 angstrom cryo-EM structure of the yeast THO center dot Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2.
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A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1
Journal of Biological Chemistry. 2021. Boughton, AJ et al. Univ Maryland, Ctr Biomol Struct & Org, Dept Chem & Biochem, College Pk, MD 20742 USA.
ABSTRACT: The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors.
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Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins
Journal of Proteome Research. 2021. Ursem, R et al. Univ Amsterdam, Dept Mol Biol & Microbial Food Safety, NL-1098 XH Amsterdam, Netherlands.
ABSTRACT: The resistance properties of the bacterial spores are partially due to spore surface proteins, similar to 30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis of the proteinaceous component of the spore coat, we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods.
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The Odorant-Binding Proteins of the Spider Mite Tetranychus urticae
International Journal of Molecular Sciences. 2021. Zhu, J et al. Austrian Inst Technol GmbH, Biosensor Technol, Konrad Lorenz Str 24, A-3430 Tulln, Austria.
ABSTRACT: Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T.
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Redox-sensitive CDC-42 clustering promotes wound closure in C. elegans
Cell reports. 2021. Xu, JX et al. Zhejiang Univ, Ctr Stem Cell & Regenerat Med, Sch Med, Affiliated Hosp 2, Hangzhou 310058, Peoples R China.
ABSTRACT: Tissue damage induces immediate-early signals, activating Rho small GTPases to trigger actin polymerization essential for later wound repair. However, how tissue damage is sensed to activate Rho small GTPases locally remains elusive. Here, we found that wounding the C. elegans epidermis induces rapid relocalization of CDC-42 into plasma membrane-associated clusters, which subsequently recruits WASP/WSP-1 to trigger actin polymerization to close the wound. In addition, wounding induces a local transient increase and subsequent reduction of H2O2, which negatively regulates the clustering of CDC-42 and wound closure.
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HSP-90/kinase complexes are stabilized by the large PPIase FKB-6
Scientific Reports. 2021. Sima, S et al. Tech Univ Munich, Ctr Integrated Prot Res, Dept Chem, Lichtenbergstr 4, D-85748 Garching, Germany.
ABSTRACT: Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase center dot Hsp90 center dot Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex.
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Unraveling the surface glycoprotein interaction network by integrating chemical crosslinking with MS-based proteomics
Chemical Science. 2021. Sun, FX et al. Georgia Inst Technol, Sch Chem & Biochem, Atlanta, GA 30332 USA.
ABSTRACT: The cell plasma membrane provides a highly interactive platform for the information transfer between the inside and outside of cells. The surface glycoprotein interaction network is extremely important in many extracellular events, and aberrant protein interactions are closely correlated with various diseases including cancer. Comprehensive analysis of cell surface protein interactions will deepen our understanding of the collaborations among surface proteins to regulate cellular activity. In this work, we developed a method integrating chemical crosslinking, an enzymatic reaction, and MS-based proteomics to systematically characterize proteins interacting with surface glycoproteins, and then constructed the surfaceome interaction network.
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Binding of cytochrome P450 27C1, a retinoid desaturase, to its accessory protein adrenodoxin
Archives of Biochemistry and Biophysics. 2021. Glass, Sarah M et al. Department of Biochemistry, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, Tennessee, 37232-0146, United States.
ABSTRACT: Of the 57 human cytochrome P450 (P450) enzymes, seven are mitochondrial: 11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1. Mitochondrial P450s utilize an electron transport system with adrenodoxin (Adx) and NADPH-adrenodoxin reductase (AdR). AdR reduces Adx, which then transfers electrons to the P450. The interactions between proteins in the mitochondrial P450 system are largely driven by electrostatic interactions, though the specifics vary depending on the P450. Unlike other mitochondrial P450s, the interaction between P450 27C1, a retinoid 3,4-desaturase expressed in the skin, and Adx remains largely uncharacterized.
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A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity
Scientific Reports. 2021. Amigues, B et al. Aix Marseille Univ AMU, CNRS, Architecture & Fonct Macromol Biol AFMB, UMR 6098, Campus Luminy,Case 932, F-13288 Marseille 09, France.
ABSTRACT: Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts.
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Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein
Structure. 2021. Ludzia, P et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4.
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A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking
Journal of Proteomics. 2021. de Jong, L et al. Univ Amsterdam, Swammerdam Inst Life Sci, Mass Spectrometry Biomol, Sci Pk 904, NL-1098 XH Amsterdam, Netherlands.
ABSTRACT: In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at proteome-wide scale and at peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 to identify cross-links induced in exponentially growing Bacillus subtilis cells treated in culture with bis(succinimidyl)-3-azidomethyl-glutarate (BAMG).
[more...]
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Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis
Analytical and Bioanalytical Chemistry. 2021. Luis Javier González et al. Ctr Genet Engn & Biotechnol CIGB, Dept Prote, Ave 31,E 158 & 190, Havana 10600, Cuba; Ctr Genet Engn & Biotechnol CIGB, Anim Biotechnol Dept, Ave 31,E 158 & 190, Havana 10600, Cuba
ABSTRACT: A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys(1)pP0 and p64K-beta Ala(1)pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0.
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Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90
Scientific reports. 2021. Schmauder, Lukas et al. Department of Chemistry, Center for Integrated Protein Research, Technische Universität München, Lichtenbergstr. 4, 85748, Garching, Germany
ABSTRACT: The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C.
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Accurate Retention Time Prediction Based on Monolinked Peptide Information to Confidently Identify Cross-Linked Peptides
Journal of the American Society for Mass Spectrometry. 2021. Huang, R et al. ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China
ABSTRACT: Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome- wide level mainly due to the computation complexity (O (n(2))) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately.
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Spontaneous protein--protein crosslinking at glutamine and glutamic acid residues in long-lived proteins
Biochemical Journal. 2021. Friedrich, MG et al. Univ Wollongong, Illawarra Hlth & Med Res Inst, Wollongong, NSW 2522, Australia.
ABSTRACT: Long-lived proteins (LLPs) are susceptible to the accumulation of both enzymatic and spontaneous post-translational modifications (PTMs). A prominent PTM observed in LLPs is covalent protein-protein crosslinking. In this study, we examined aged human lenses and found several proteins to be crosslinked at Glu and Gln residues. This new covalent bond involves the amino group of Lys or an alpha-amino group. A number of these crosslinks were found in intermediate filament proteins. Such crosslinks could be reproduced experimentally by incubation of Glu- or Gln-containing peptides and their formation was consistent with an amino group attacking a glutarimide intermediate.
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Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain
PNAS. 2021. Hevler, JF et al. Univ Utrecht, Utrecht Inst Pharmaceut Sci, Bijvoet Ctr Biomol Res, Biomol Mass Spectrometry & Prote, NL-3584 CH Utrecht, Netherlands.
ABSTRACT: Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with similar to 10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM1(2) complex (PDBDEV_00000092).
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The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein
Nature communications. 2021. Lu, S et al. Univ Calif San Diego, Dept Cellular & Mol Med, San Diego, CA 92093 USA.
ABSTRACT: The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the similar to 30kb viral RNA genome to aid its packaging into the 80-90nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates.
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Structural insights into preinitiation complex assembly on core promoters
Science. 2021. Chen, XZ et al. Fudan Univ, Shanghai Canc Ctr, Inst Biomed Sci, State Key Lab Genet Engn, Shanghai 200032, Peoples R China.
ABSTRACT: Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters).
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Structural basis of Integrator-mediated transcription regulation
Science. 2021. Fianu, I et al. Max Planck Inst Biophys Chem, Dept Mol Biol, D-37077 Gottingen, Germany.
ABSTRACT: Integrator and protein phosphatase 2A (PP2A) form a complex that dephosphorylates paused RNA polymerase II (Pol II), cleaves the nascent RNA, and terminates transcription. We report the structure of the pretermination complex containing a human Integrator-PP2A complex bound to paused Pol II. Integrator binds Pol II and the pausing factors DSIF and NELF to exclude binding of the elongation factors SPT6 and PAF1 complex. Integrator also binds the C-terminal domain of Pol II and positions PP2A to counteract Pol II phosphorylation and elongation.
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Structural basis of human transcription–DNA repair coupling
Nature. 2021. Kokic, G et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Transcription-coupled DNA repair removes bulky DNA lesions from the genome(1,2) and protects cells against ultraviolet (UV) irradiation(3). Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4(CSA) and UV-stimulated scaffold protein A (UVSSA)(3). Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6.
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DNA-driven condensation assembles the meiotic DNA break machinery
NATURE. 2021. Bouuaert, CC et al. Mem Sloan Kettering Canc Ctr, Program Mol Biol, New York, NY 10021 USA.
ABSTRACT: The accurate segregation of chromosomes during meiosis-which is critical for genome stability across sexual cycles-relies on homologous recombination initiated by DNA double-strand breaks (DSBs) made by the Spo11 protein(1,2). The formation of DSBs is regulated and tied to the elaboration of large-scale chromosome structures(3-5), but the protein assemblies that execute and control DNA breakage are poorly understood. Here we address this through the molecular characterization of Saccharomyces cerevisiae RMM (Rec114, Mei4 and Mer2) proteins-essential, conserved components of the DSB machinery(2).
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The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1
Nature Structural & Molecular Biology. 2021. Lupton, CJ et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic, Australia.
ABSTRACT: Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human similar to 640-kDa NF1 homodimer features a gigantic 30 x 10 nm array of alpha-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation.
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Mapping protein interactions in the active TOM-TIM23 supercomplex
Nature Communications. 2021. Gomkale, R et al. Univ Med Ctr Gottingen, Dept Cellular Biochem, Gottingen, Germany.
ABSTRACT: Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs.
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Structural and functional characterization of the Spo11 core complex
nature structural & molecular biology. 2021. Bouuaert, CC et al. Mem Sloan Kettering Canc Ctr, Mol Biol Program, 1275 York Ave, New York, NY 10021 USA.
ABSTRACT: Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomycescerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation.
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Structural insights into how Prp5 proofreads the pre-mRNA branch site
Nature. 2021. Zhang, ZW et al. MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.
ABSTRACT: During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. (1-4)). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism(5). Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed.
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Accurate and automated high-coverage identification of chemically cross-linked peptides with MaxLynx
Analytical chemistry. 2021. Yilmaz, S et al. Max Planck Inst Biochem, Computat Syst Biochem Res Grp, D-82152 Martinsried, Germany; Univ Bergen, Dept Biol & Med Psychol, N-5007 Bergen, Norway
ABSTRACT: Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine.
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The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II
Journal of Biological Chemistry. 2021. Osman, S et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro.
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Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ
Nature communications. 2021. Chen, JH et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol Natl Ctr Prot Sci,Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS.
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Molecular basis of F-actin regulation and sarcomere assembly via myotilin
PLoS biology. 2021. Kostan, J et al. Univ Vienna, Dept Struct & Computat Biol, Max Perutz Labs, Vienna, Austria.
ABSTRACT: Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and alpha-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches.
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Cross‐linking mass spectrometry reveals the structural topology of peripheral NuRD subunits relative to the core complex
The Federation of European Biochemical Societies Journal. 2021. Spruijt, CG et al. Radboud Univ Nijmegen, Oncode Inst, Radboud Inst Mol Life Sci, Fac Sci,Dept Mol Biol, NL-6525 GA Nijmegen, Netherlands.
ABSTRACT: The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported.
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Identification of protein direct interactome with genetic code expansion and search engine OpenUaa
Advanced Biology. 2021. Liu, C et al. Beihang Univ, Sch Med & Engn, Beijing Adv Innovat Ctr Big Data Based Precis Med, Beijing 100191, Peoples R China; Beihang Univ, Key Lab Big Data Based Precis Med, Minist Ind & Informat Technol, Beijing 100191, Peoples R China; Zhejiang Univ, MOE Lab Biosyst Homeostasis & Protect, Hangzhou 310058, Peoples R China; Zhejiang Univ, Life Sci Inst, Hangzhou 310058, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA; Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA; Chinese Acad Agr Sci, Inst Anim Sci, Beijing 100193, Peoples R China
ABSTRACT: Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage.
[more...]
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Membrane interactions of α-Synuclein revealed by multiscale molecular dynamics simulations, Markov state models, and NMR
JOURNAL OF PHYSICAL CHEMISTRY B. 2021. Amos, SBTA et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: alpha-Synuclein (alpha S) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of alpha S to membranes is a likely first step in the molecular pathophysiology of PD. The alpha S molecule can adopt multiple conformations, being largely disordered in water, adopting a beta-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of alpha-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces.
[more...]
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Elucidating the molecular mechanism of dynamic photodamage of photosystem II membrane protein complex by integrated proteomics strategy
CCS Chemistry. 2021. Zhou, Y et al. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023
ABSTRACT: Photosystem II (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex on the thylakoid membrane, is a primary target of light-induced photodamage. However, the overall molecular details of the conformation and composition dynamics of PSII photodamage are still controversial. In this study, we investigated systematically the dynamic conformation, degradation, and oxidation processes of PSII photodamage by integrating chemical cross-linking and top-down proteomics strategies.
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Colicin-mediated transport of DNA through the iron transporter FepA
MBio. 2021. Cohen-Khait, R et al. Univ Oxford, Dept Biochem, Oxford, England
ABSTRACT: Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria.
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TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival
The Journal of biological chemistry. 2021. Scheer, Elisabeth et al. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique, UMR7104, Institut National de la Santé et de la Recherche Médicale, U964, Université de Strasbourg, Illkirch, France
ABSTRACT: The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled invitro constituting a building block for the further assembly of either lobe A or B in TFIID.
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Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM
PNAS. 2021. Zheng, W et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol,Natl Ctr Prot Sci Shanghai, Shanghai 200031, Peoples R China.
ABSTRACT: The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-angstrom resolution.
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Structural analysis of the PTEN: P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1
Science Signaling. 2021. D'Andrea, L et al. Monash Univ, Biomed Discovery Inst, Clayton, Vic 3800, Australia; Monash Univ, Monash Inst Pharmaceut Sci, Drug Discovery Biol Theme, Parkville, Vic 3052, Australia
ABSTRACT: The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P-3 to PI(4,5)P-2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by G beta gamma subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P-3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation.
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Solution structure and conformational flexibility of a polyketide synthase module
JACS. 2021. Klaus, M et al. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue Strasse 15, Frankfurt am Main 60438, Germany
ABSTRACT: Polyketide synthases (PKSs) are versatile C–C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains—well characterized on their own—are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs.
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NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins
Nature communications. 2021. Scheu, AHA et al. Univ Oxford, Dept Biochem, South Parks Rd, Oxford OX1 3QU, England.
ABSTRACT: The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex.
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Carboxypeptidase Y Assisted Disulfide-Bond Identification with Linearized Database Search
Analytical Chemistry. 2021. Qiang, Jiali et al. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 100 Haike Road, Pudong, Shanghai 201210, China
ABSTRACT: A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)".
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SAGA and SAGA-like SLIK transcriptional coactivators are structurally and biochemically equivalent
Journal of Biological Chemistry. 2021. Adamus, K et al. Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic, Australia.
ABSTRACT: The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGAsubunit, causing loss of Spt8, one of the subunits that interacts with the TATA-binding protein (TBP). SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast, and both SAGA and SLIK perform chromatin modifications. The two complexes have been speculated to uniquely contribute to transcriptional regulation, but their respective contributions are not clear.
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Characterization of the Fc–III–4C-based recombinant protein expression system by using carbonic anhydrase as the model protein
Protein Expression and Purification. 2021. Gong, YY et al. Tsinghua Univ, Ctr Synthet & Systemat Biol, Sch Life Sci, MOE Key Lab Bioinformat, Beijing 100084, Peoples R China.
ABSTRACT: Development of new affinity tags is important for recombinant protein expression and purification. Based on our earlier work, we devised an affinity tag by addition of two cysteine residues onto the N- and C-termini of the Fc-III peptide and designated as the Fc-III-4C tag, in which four cysteine residues form two disulfide linkages. The binding affinity of Fc-III-4C tag to human IgG is measured as 2.28 nM (K-d) and is 100 times higher than that of the Fc-III tag to IgG. Fc-III-4C tagged carbonic anhydrase (CA) can be effectively purified with IgG-immobilized beads, and Fc-III-4C tag does not possess adverse effects on the structure and stability of CA.
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Elucidating the Molecular Mechanism of Dynamic Photodamage of Photosystem II Membrane Protein Complex by Integrated Proteomics Strategy
CCS Chemistry. 2021. Zhou, Y et al. CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023
ABSTRACT: Photosystem II (PSII), as a multiple-subunit chloroplast membrane-associated pigment-protein complex on the thylakoid membrane, is a primary target of light-induced photodamage. However, the overall molecular details of the conformation and composition dynamics of PSII photodamage are still controversial. In this study, we investigated systematically the dynamic conformation, degradation, and oxidation processes of PSII photodamage by integrating chemical cross-linking and top-down proteomics strategies.
[more...]
Use: pLink



Cryo-EM of mammalian PA28$\alpha$$\beta$-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28$\alpha$$\beta$
Nature communications. 2021. Chen, JH et al. Chinese Acad Sci, Ctr Excellence Mol Cell Sci, Shanghai Inst Biochem & Cell Biol, State Key Lab Mol Biol Natl Ctr Prot Sci,Shanghai, Shanghai 200031, Peoples R China
ABSTRACT: The proteasome activator PA28 alpha beta affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28 alpha beta -iCP structure, how PA28 alpha beta regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28 alpha beta -iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28 alpha beta and iCP through XL-MS.
[more...]
Use: pLink



Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR
JOURNAL OF PHYSICAL CHEMISTRY B. 2021. Amos, SBTA et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: alpha-Synuclein (alpha S) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of alpha S to membranes is a likely first step in the molecular pathophysiology of PD. The alpha S molecule can adopt multiple conformations, being largely disordered in water, adopting a beta-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of alpha-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces.
[more...]
Use: pLink; pParse



Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa
Advanced Biology. 2021. Liu, C et al. Beihang Univ, Sch Med & Engn, Beijing Adv Innovat Ctr Big Data Based Precis Med, Beijing 100191, Peoples R China; Beihang Univ, Key Lab Big Data Based Precis Med, Minist Ind & Informat Technol, Beijing 100191, Peoples R China; Zhejiang Univ, MOE Lab Biosyst Homeostasis & Protect, Hangzhou 310058, Peoples R China; Zhejiang Univ, Life Sci Inst, Hangzhou 310058, Peoples R China; Tsinghua Univ, Tsinghua Inst Multidisciplinary Biomed Res, Beijing 102206, Peoples R China; Natl Inst Biol Sci NIBS, Beijing 102206, Peoples R China; Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA; Univ Calif San Francisco, Cardiovasc Res Inst, San Francisco, CA 94158 USA; Chinese Acad Agr Sci, Inst Anim Sci, Beijing 100193, Peoples R China
ABSTRACT: Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage.
[more...]
Use: pLink



Solution Structure and Conformational Flexibility of a Polyketide Synthase Module
JACS. 2021. Klaus, M et al. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue Strasse 15, Frankfurt am Main 60438, Germany
ABSTRACT: Polyketide synthases (PKSs) are versatile C–C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties. Particularly, the domains—well characterized on their own—are poorly understood in their arrangement, conformational dynamics, and interplay in the intricate quaternary structure of modular PKSs.
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Use: pLink



Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2021. Zhang, H et al. Peking Univ, Synthet & Funct Biomol Ctr, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Beijing 100871, Peoples R China.
ABSTRACT: The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells.
[more...]
Use: pLink



Cysteine inducing formation and reshuffling of disulfide bonds in cold-extruded whey protein molecules: From structural and functional characteristics to cytotoxicity
Food Chemistry. 2021. Yang, N et al. Northeast Agr Univ, Coll Food Sci & Technol, Key Lab Dairy Sci, Minist Educ, Harbin 150030, Heilongjiang, Peoples R China.
ABSTRACT: Polymer chemistry, rheology and cytotoxicity of cysteine initiated S-S redistribution in cold-extruded whey protein (TWPI) molecules were investigated. The locations of disulfide bonds in whey protein isolate (WPI), WPI dried without being extruded (OWPI) and cold-extruded WPI (TWPI), Cysteine (Cys)-treated WPI (WPI-Cys), OWPI (OWPI-Cys) and TWPI (TWPI-Cys) were precisely analyzed using liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software approaches.
[more...]
Use: pLink



MS Annika: A New Cross-Linking Search Engine
Journal of Proteome Research. 2021. Pirklbauer, GJ et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct.
[more...]
Use: pLink



Structure of cyanobacterial phycobilisome core revealed by structural modeling and chemical cross-linking
Science Advances. 2021. Liu, HJ et al. Washington Univ, Dept Chem, St Louis, MO 63130 USA.
ABSTRACT: In cyanobacteria and red algae, the structural basis dictating efficient excitation energy transfer from the phycobilisome (PBS) antenna complex to the reaction centers remains unclear. The PBS has several peripheral rods and a central core that binds to the thylakoid membrane, allowing energy coupling with photosystem II (PSII) and PSI. Here, we have combined chemical cross-linking mass spectrometry with homology modeling to propose a tricylindrical cyanobacterial PBS core structure. Our model reveals a side-view crossover configuration of the two basal cylinders, consolidating the essential roles of the anchoring domains composed of the ApcE PB loop and ApcD, which facilitate the energy transfer to PSII and PSI, respectively.
[more...]
Use: pLink



Clostridium perfringens suppressing activity in black soldier fly protein preparations
LWT - Food Science and Technology. 2021. Dong, LY et al. Wageningen Res, Food & Biobased Res, Bornse Weilanden 9, NL-6708 WG Wageningen, Netherlands.
ABSTRACT: Clostridium perfringens is a commensal, but also an opportunistic pathogen that can lead to lethal diseases as a result of overgrowth when homeostasis is disrupted. The current course of treatment is antibiotics. However, with increasing antibiotic resistance alternatives are required. We investigated the antimicrobial capacity of digest from different black soldier fly- and mealworm-derived fractions towards C. perfringens by using in vitro models. Culturing C. perfringens with digest of insect-derived fractions showed that fractions containing black soldier fly larvae protein significantly (p < 0.05) inhibited the growth of C.
[more...]
Use: pNovo



Peptide presentations of marsupial MHC class I visualize immune features of lower mammals paralleled with bats
JOURNAL OF IMMUNOLOGY. 2021. Wang, PY et al. Wenzhou Med Univ, Sch Lab Med & Life Sci, Wenzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 1, Sch Med, State Key Lab Diag & Treatment Infect Dis, Hangzhou, Zhejiang, Peoples R China; Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, 155 Changbai Rd, Beijing 100101, Peoples R China
ABSTRACT: Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus -host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex.
[more...]
Use: pNovo



Improved Identification of Small Open Reading Frames Encoded Peptides by Top-Down Proteomic Approaches and De Novo Sequencing
International journal of molecular sciences. 2021. Wang, B et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, 152 Luoyu Rd, Wuhan 430079, Peoples R China.
ABSTRACT: Small open reading frames (sORFs) have translational potential to produce peptides that play essential roles in various biological processes. Nevertheless, many sORF-encoded peptides (SEPs) are still on the prediction level. Here, we construct a strategy to analyze SEPs by combining top-down and de novo sequencing to improve SEP identification and sequence coverage. With de novo sequencing, we identified 1682 peptides mapping to 2544 human sORFs, which were all first characterized in this work. Two-thirds of these new sORFs have reading frame shifts and use a non-ATG start codon.
[more...]
Use: pNovo



Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues
Frontiers in cell and developmental biology. 2021. Pan, N et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan, Peoples R China.
ABSTRACT: Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located within the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins, especially ncRNA-encoded proteins, expressed in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies.
[more...]
Use: pNovo



Peptide Presentations of Marsupial MHC Class I Visualize Immune Features of Lower Mammals Paralleled with Bats
JOURNAL OF IMMUNOLOGY. 2021. Wang, PY et al. Wenzhou Med Univ, Sch Lab Med & Life Sci, Wenzhou, Peoples R China; Zhejiang Univ, Affiliated Hosp 1, Sch Med, State Key Lab Diag & Treatment Infect Dis, Hangzhou, Zhejiang, Peoples R China; Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, 155 Changbai Rd, Beijing 100101, Peoples R China
ABSTRACT: Marsupials are one of three major mammalian lineages that include the placental eutherians and the egg-laying monotremes. The marsupial brushtail possum is an important protected species in the Australian forest ecosystem. Molecules encoded by the MHC genes are essential mediators of adaptive immune responses in virus -host interactions. Yet, nothing is known about the peptide presentation features of any marsupial MHC class I (MHC I). This study identified a series of possum MHC I Trvu-UB*01:01 binding peptides derived from wobbly possum disease virus (WPDV), a lethal virus of both captive and feral possum populations, and unveiled the structure of marsupial peptide/MHC I complex.
[more...]
Use: pNovo



Computationally instrument-resolution-independent de novo peptide sequencing for high-resolution devices
Nature Machine Intelligence. 2021. Qiao, R et al. Univ Waterloo, Dept Stat & Actuarial Sci, Waterloo, ON, Canada.
ABSTRACT: De novo peptide sequencing is the key technology for finding novel peptides from mass spectra. The overall quality of sequencing results depends on the de novo peptide sequencing algorithm as well as the quality of mass spectra. Over the past decade, the resolution and accuracy of mass spectrometers have improved by orders of magnitude and higher-resolution mass spectra have been generated. How to effectively take advantage of those high-resolution data without substantially increasing the computational complexity remains a challenge for de novo peptide sequencing tools.
[more...]
Use: pNovo



Reaction Tracking and High-Throughput Screening of Active Compounds in Combinatorial Chemistry by Tandem Mass Spectrometry Molecular Networking
Analytical chemistry. 2021. Chung, HH et al. Natl Taiwan Univ, Dept Chem, Taipei 10617, Taiwan.
ABSTRACT: Combinatorial synthesis has been widely used as an efficient strategy to screen for active compounds. Mass spectrometry is the method of choice in the identification of hits resulting from high-throughput screenings due to its high sensitivity, specificity, and speed. However, manual data processing of mass spectrometry data, especially for structurally diverse products in combinatorial chemistry, is extremely time-consuming and one of the bottlenecks in this process. In this study, we demonstrated the effectiveness of a tandem mass spectrometry molecular networking-based strategy for product identification, reaction dynamics monitoring, and active compound targeting in combinatorial synthesis.
[more...]
Use: pNovo



Enhancing open modification searches via a combined approach facilitated by Ursgal
Journal of Proteome Research. 2021. Schulze, S et al. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT: The identification of peptide sequences and their posttranslational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs).
[more...]
Use: pGlyco



StrucGP: de novo structural sequencing of site-specific N-glycan on glycoproteins using a modularization strategy
Nature Methods. 2021. Shen, JC et al. Northwest Univ, Coll Life Sci, Xian, Peoples R China.
ABSTRACT: Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes.
[more...]
Use: pGlyco



In-depth site-specific analysis of N-glycoproteome in human cerebrospinal fluid and glycosylation landscape changes in Alzheimer's disease
Molecular & Cellular Proteomics. 2021. Chen, ZW et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF.
[more...]
Use: pGlyco



Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: Implications for vaccination and antibody therapeutics
Glycobiology. 2021. Zhou, DP et al. Tongji Univ, Sch Med, 1239 Siping Rd, Shanghai 200092, Peoples R China.
ABSTRACT: Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis.
[more...]
Use: pGlyco



Fecal multi-omics analysis reveals diverse molecular alterations of gut ecosystem in COVID-19 patients
ANALYTICA CHIMICA ACTA. 2021. He, FX et al. Sun Yat Sen Univ, Guangdong Prov Key Lab Biomed Imaging, Affiliated Hosp 5, Zhuhai 519000, Peoples R China.
ABSTRACT: Gut ecosystem has profound effects on host physiology and health. Gastrointestinal (GI) symptoms were frequently observed in patients with COVID-19. Compared with other organs, gut antiviral response can result in more complicated immune responses because of the interactions between the gut microbiota and host immunity. However, there are still large knowledge gaps in the impact of COVID-19 on gut molecular profiles and commensal microbiome, hindering our comprehensive understanding of the pathogenesis of SARS-CoV-2 and the treatment of COVID-19.
[more...]
Use: pGlyco



Sorbitol is a severity biomarker for PMM2-CDG with therapeutic implications
Annals of Neurology. 2021. Ligezka, AN et al. Mayo Clin, Dept Clin Genom, 200 First St SW, Rochester, MN 55905 USA.
ABSTRACT: Objective Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG. Methods We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment.
[more...]
Use: pGlyco



Glycoproteomics analysis reveals differential expression of site-specific glycosylation in human milk whey during lactation
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2021. Wang, ZY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Protein N-glycosylation in human milk whey plays a substantial role in infant health during postnatal development. Changes in site-specific glycans in milk whey reflect the needs of infants under different circumstances. However, the conventional glycoproteomics analysis of milk whey cannot reveal the changes in site-specific glycans because the attached glycans are typically enzymatically removed from the glycoproteins prior to analysis. In this study, N-glycoproteomics analysis of milk whey was performed without removing the attached glycans, and 330 and 327 intact glycopeptides were identified in colostrum and mature milk whey, respectively.
[more...]
Use: pQuant; pGlyco



Automated intact glycopeptide enrichment method facilitating highly reproducible analysis of serum site-specific N-glycoproteome
Analytical Chemistry. 2021. Liu, LY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min.
[more...]
Use: pGlyco



Evaluation and Optimization of High-Field Asymmetric Waveform Ion-Mobility Spectrometry for Multiplexed Quantitative Site-Specific N-Glycoproteomics
Analytical Chemistry. 2021. Fang, P et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.
ABSTRACT: The heterogeneity and complexity of glycosylation hinder the depth of site-specific glycoproteomics analysis. High-field asymmetric-waveform ion-mobility spectrometry (FAIMS) has been shown to improve the scope of bottom-up proteomics. The benefits of FAIMS for quantitative N-glycoproteomics have not been investigated yet. In this work, we optimized FAIMS settings for N-glycopeptide identification, with or without the tandem mass tag (TMT) label. The optimized FAIMS approach significantly increased the identification of site-specific N-glycopeptides derived from the purified immunoglobulin M (IgM) protein or human lymphoma cells.
[more...]
Use: pGlyco; pQuant



The need for community standards to enable accurate comparison of glycoproteomics algorithm performance
molecules. 2021. Hackett, WE et al. Boston Univ, Bioinformat Program, Boston, MA 02215 USA.
ABSTRACT: Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco.
[more...]
Use: pGlyco



Extensive heterogeneity of glycopeptides in plasma revealed by deep glycoproteomic analysis using size-exclusion chromatography
Molecular Omics. 2021. Saraswat, M et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT: Several plasma glycoproteins are clinically useful as biomarkers in a variety of diseases. Although thousands of proteins are present in plasma, >95% of the plasma proteome by mass is represented by only 22 proteins. This necessitates strategies to deplete the abundant proteins and enrich other subsets of proteins. Although glycoproteins are abundant in plasma, in routine proteomic analyses, glycopeptides are not often investigated. Traditional methods such as lectin-based enrichment of glycopeptides followed by deglycosylation have helped understand the glycoproteome, but they lack any information about the attached glycans.
[more...]
Use: pGlyco



Mass spectrometry analysis of SARS-CoV-2 nucleocapsid protein reveals camouflaging glycans and unique post-translational modifications
Infectious Microbes & Diseases. 2021. Sun, ZY et al. State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, No.79 Qingchun Road, Shangcheng District, Hangzhou 310003, China
ABSTRACT: The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion.
[more...]
Use: pGlyco



O-GlcNAcylation of MEK2 promotes the proliferation and migration of breast cancer cells
Glycobiology. 2021. Xu, YY et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Mitogen-activated protein kinase kinases are an important part of evolutionary conserved signaling modules that are involved in a variety of cellular processes in response to environmental stimuli. Among them, mitogen-activated protein kinase kinase 2 (MEK2) is the most crucial upstream signaling pathway of ERK1/2 cascade as a therapeutic target for overcoming Ras-driven cancers. However, the mechanisms of MEK2 regulation during tumor progression remain not fully elucidated. Herein, we identified that MEK2 was post-translationally regulated by O-GlcNAcylation.
[more...]
Use: pGlyco



Enhancing open modification searches via a combined approach facilitated by ursgal
Journal of Proteome Research. 2021. Schulze, S et al. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT: The identification of peptide sequences and their posttranslational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs).
[more...]
Use: pGlyco



Identification of dysregulated complement activation pathways driven by N-glycosylation alterations in T2D patients
frontiers in Chemistry. 2021. Zhao, Y et al. Natl Inst Metrol, Ctr Adv Measurement Sci, Beijing, Peoples R China.
ABSTRACT: Diabetes has become a major public health concern worldwide, most of which are type 2 diabetes (T2D). The diagnosis of T2D is commonly based on plasma glucose levels, and there are no reliable clinical biomarkers available for early detection. Recent advances in proteome technologies offer new opportunity for the understanding of T2D; however, the underlying proteomic characteristics of T2D have not been thoroughly investigated yet. Here, using proteomic and glycoproteomic profiling, we provided a comprehensive landscape of molecular alterations in the fasting plasma of the 24 Chinese participants, including eight T2D patients, eight prediabetic (PDB) subjects, and eight healthy control (HC) individuals.
[more...]
Use: pGlyco



Quantification of intact O-Glycopeptides on haptoglobin in sera of patients with hepatocellular carcinoma and liver cirrhosis
frontiers in Chemistry. 2021. Shu, H et al. Fudan Univ, Zhongshan Hosp, Liver Canc Inst, Shanghai, Peoples R China.
ABSTRACT: Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC.
[more...]
Use: pGlyco; pQuant



Precision N-glycoproteomic profiling of murine peritoneal macrophages after different stimulations
Frontiers in Immunology. 2021. Yang, LJ et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT: Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis.
[more...]
Use: pGlyco



Dissection of the Glycosylation in the Biosynthesis of the Heptadecaglycoside Antibiotic Saccharomicin A
JOURNAL OF ORGANIC CHEMISTRY. 2021. Zhao, JF et al. Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT: Oligosaccharide natural products have diverse biological activities and represent a potentially important source for drug development. In this study, we focus on the glycosylation pathway in the biosynthesis of saccharomicin A (SA-A), an oligosaccharide antibiotic containing 17 sugar moieties. By extensive gene-knockout studies with comparative metabolic profile analysis, we established a complete pathway in assembling the heptadecasaccharide chain of SA-A, the longest saccharide chain found in natural products.
Use: pGlyco



Effective enrichment strategy using boronic acid-functionalized mesoporous graphene--silica composites for intact N-and O-linked glycopeptide analysis in human serum
Analytical Chemistry. 2021. Kong, Siyuan et al. Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai 200032, Peoples R China; Fudan Univ, Peoples Hosp 5, Shanghai 200032, Peoples R China; Fudan Univ, Shanghai Key Lab Med Epigenet, Int Colab Med Epigenet & Metab, Minist Sci & Technol,Inst Biomed Sci, Shanghai 200032, Peoples R China
ABSTRACT: The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO(2)-GLYMO-APB) for isolating intact glycopeptides from complex biological samples.
[more...]
Use: pGlyco



GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control
Nature Communications. 2021. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.
ABSTRACT: Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification. Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics.
[more...]
Use: pGlyco



Precision N-Glycoproteomic Profiling of Murine Peritoneal Macrophages After Different Stimulations
Frontiers in Immunology. 2021. Yang, LJ et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT: Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis.
[more...]
Use: pGlyco



Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications
Infectious Microbes & Diseases. 2021. Sun, ZY et al. State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, No.79 Qingchun Road, Shangcheng District, Hangzhou 310003, China
ABSTRACT: The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion.
[more...]
Use: pGlyco



In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease
Molecular & Cellular Proteomics. 2021. Chen, ZW et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF.
[more...]
Use: pGlyco



Identification of Dysregulated Complement Activation Pathways Driven by N-Glycosylation Alterations in T2D Patients
frontiers in Chemistry. 2021. Zhao, Y et al. Natl Inst Metrol, Ctr Adv Measurement Sci, Beijing, Peoples R China.
ABSTRACT: Diabetes has become a major public health concern worldwide, most of which are type 2 diabetes (T2D). The diagnosis of T2D is commonly based on plasma glucose levels, and there are no reliable clinical biomarkers available for early detection. Recent advances in proteome technologies offer new opportunity for the understanding of T2D; however, the underlying proteomic characteristics of T2D have not been thoroughly investigated yet. Here, using proteomic and glycoproteomic profiling, we provided a comprehensive landscape of molecular alterations in the fasting plasma of the 24 Chinese participants, including eight T2D patients, eight prediabetic (PDB) subjects, and eight healthy control (HC) individuals.
[more...]
Use: pGlyco



Glycoproteomics Analysis Reveals Differential Expression of Site-Specific Glycosylation in Human Milk Whey during Lactation
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2021. Wang, ZY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Protein N-glycosylation in human milk whey plays a substantial role in infant health during postnatal development. Changes in site-specific glycans in milk whey reflect the needs of infants under different circumstances. However, the conventional glycoproteomics analysis of milk whey cannot reveal the changes in site-specific glycans because the attached glycans are typically enzymatically removed from the glycoproteins prior to analysis. In this study, N-glycoproteomics analysis of milk whey was performed without removing the attached glycans, and 330 and 327 intact glycopeptides were identified in colostrum and mature milk whey, respectively.
[more...]
Use: pQuant; pGlyco



Analysis of Serum Paraoxonase 1 Using Mass Spectrometry and Lectin Immunoassay in Patients With Alpha-Fetoprotein Negative Hepatocellular Carcinoma
FRONTIERS IN ONCOLOGY. 2021. Cao, XY et al. Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT: The diagnosis of AFP (alpha-fetoprotein)-negative HCC (hepatocellular carcinoma) mostly relies on imaging and pathological examinations, and it lacks valuable and practical markers. Protein N-glycosylation is a crucial post-translation modifying process related to many biological functions in an organism. Alteration of N-glycosylation correlates with inflammatory diseases and infectious diseases including hepatocellular carcinoma. Here, serum N-linked intact glycopeptides with molecular weight (MW) of 40-55 kDa were analyzed in a discovery set (n = 40) including AFP-negative HCC and liver cirrhosis (LC) patients using label-free quantification methodology.
[more...]
Use: pGlyco; pQuant



Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome
Analytical Chemistry. 2021. Liu, LY et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min.
[more...]
Use: pGlyco



Effective Enrichment Strategy Using Boronic Acid-Functionalized Mesoporous Graphene--Silica Composites for Intact N-and O-Linked Glycopeptide Analysis in Human Serum
Analytical Chemistry. 2021. Kong, SY et al. Fudan Univ, Peoples Hosp 5, Shanghai 200032, Peoples R China.
ABSTRACT: The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO(2)-GLYMO-APB) for isolating intact glycopeptides from complex biological samples.
[more...]
Use: pGlyco



Enhancing Open Modification Searches via a Combined Approach Facilitated by Ursgal
Journal of Proteome Research. 2021. Schulze, S et al. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT: The identification of peptide sequences and their posttranslational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs).
[more...]
Use: pGlyco



Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression
Science Advances. 2021. Li, SS et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China.
ABSTRACT: Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain.
[more...]
Use: pDeep



DeepPhospho accelerates DIA phosphoproteome profiling through in silico library generation
Nature Communications. 2021. Lou, RH et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China; ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China; ShanghaiTech Univ, Sch Informat Sci & Technol, Shanghai 201210, Peoples R China; Shanghai Engn Res Ctr Intelligent Vis & Imaging, Shanghai 201210, Peoples R China
ABSTRACT: The coverage and throughput of data-independent acquisition (DIA)-based phosphoproteomics is limited by its dependence on experimental spectral libraries. Here the authors develop a DIA workflow based on in silico spectral libraries generated by a novel deep neural network to expand phosphoproteome coverage.Phosphoproteomics integrating data-independent acquisition (DIA) enables deep phosphoproteome profiling with improved quantification reproducibility and accuracy compared to data-dependent acquisition (DDA)-based phosphoproteomics.
[more...]
Use: pDeep



Structural basis of human transcription-DNA repair coupling
Nature. 2021. Kokic, G et al. MPI
ABSTRACT: The unbounded permutations of biological molecules, includingproteinsand their constituent peptides, present a dilemma in identifying thecomponents of complex biosamples. Sequence search algorithms usedto identify peptide spectra can be expanded to cover larger classesof molecules, including more modifications, isoforms, and atypicalcleavage, but at the cost of false positives or false negatives dueto the simplified spectra they compute from sequence records. Spectrallibrary searching can help solve this issue by precisely matchingexperimental spectra to library spectra with excellent sensitivityand specificity.
[more...]
Use: pLink




2020




Characterization of lysine monomethylome and methyltransferase in model cyanobacterium Synechocystis sp. PCC 6803
Genomics, proteomics & bioinformatics. 2020. Lin, XH et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT: Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp.
[more...]
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An algorithm to improve the speed of Semi and non-specific enzyme searches in proteomics
Current bioinformatics. 2020. Rolfs, Z et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time-consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and nonspecific enzyme searches and could be applied to existing search programs. Methods: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once.
[more...]
Use: pFind



Protocol for proximity-dependent proteomic profiling in yeast cells by APEX and Alk-Ph probe
STAR Protocols. 2020. Li, Yi et al. Peking University
ABSTRACT: Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome.
[more...]
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Protocol for proximity-dependent proteomic profiling in yeast cells by APEX and Alk-Ph probe
STAR Protocols. 2020. Li, Yi et al. Peking University
ABSTRACT: Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome.
[more...]
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DPHL: a DIA pan-human protein mass spectrometry library for robust biomarker discovery
Genomics, proteomics & bioinformatics. 2020. Zhu, Tiansheng et al. Westlake Univ, Zhejiang Prov Lab Life Sci & Biomed, Hangzhou 310024, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Peoples R China
ABSTRACT: To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel dataindependent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples.
[more...]
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Accelerated lysis and proteolytic digestion of biopsy-level fresh-frozen and FFPE tissue samples using pressure cycling technology
Journal of proteome research. 2020. Gao, Huanhuan et al. Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China
ABSTRACT: Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and high-throughput proteomic studies of both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue of biopsy scale for biomarker discovery. Here, we present an improved PCT method accelerating the conventional procedures by about two-fold without sacrificing peptide yield, digestion efficiency, peptide, and protein identification. The time required for processing 16 tissue samples from tissues to peptides is reduced from about 6 to about 3 h.
[more...]
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Large-scale identification of N-linked intact glycopeptides in human serum using HILIC enrichment and spectral library search
Molecular & cellular proteomics : MCP. 2020. Shu, Qingbo et al. Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences
ABSTRACT: Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control.
[more...]
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Global chemical modifications comparison of human plasma proteome from two different age groups
Scientific Reports. 2020. Liu, Yongtao et al. Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing, Peoples R China
ABSTRACT: In this study, two groups of human plasma proteome at different age groups (old and young) were used to perform a comparison of global chemical modifications, as determined by tandem mass spectrometry (MS/MS) combined with non-limiting modification identification algorithms. The sulfhydryl in the cysteine A total of 4 molecular modifications were found to have significant differences passing random grouping tests: the succinylation and phosphorylation modification of cysteine (Cys, C) and the modification of lysine (Lys, K) with threonine (Thr, T) were significantly higher in the old group than in the young group, while the carbamylation of lysine was lower in the young group.
[more...]
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An innovative artificial photosystem II constructed from PSII core of Thermosynechococcus vulcanus and LHCII of Pisum sativum-A new approach for studying the function of photosynthetic antenna
Plant Physiology and Biochemistry. 2020. Li, Ling et al. Chinese Acad Sci, Inst Bot, Key Lab Plant Resources, Beijing Bot Garden, Beijing 100093, Peoples R China; Chinese Acad Sci, Qingdao Inst, Shanghai Inst Technol Phys, Binhai Rd 50, Qingdao 264000, Peoples R China
ABSTRACT: In photosynthesis, the antenna system captures solar energy and transfers the excitations to photosystem II (PSII) core complex where charge separation, water splitting and oxygen evolution occur. In the evolution of photosynthesis from aquatic to terrestrial environments, the structure of PSII core complex was highly conserved while a variety of antenna forms became differentiated. In order to study the principles for energy transport from antenna to the PSII reaction center, we have explored whether the major light harvesting complex of PSII (LHCII) of higher plants can transfer energy to the cyanobacteria PSII core complexes (CC).
[more...]
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Identification of modified peptides using localization-aware open search
Nature communications. 2020. Yu, FC et al. Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA.
ABSTRACT: Identification of post-translationally or chemically modified peptides in mass spectrometry-based proteomics experiments is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive analysis of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-containing (shifted) and regular fragment ions are indexed and used in scoring.
[more...]
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The proteome landscape of the kingdoms of life
Nature. 2020. Muller, JB et al. Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany.
ABSTRACT: Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported(1), advances in mass-spectrometry-based proteomics(2)have enabled increasingly comprehensive identification and quantification of the human proteome(3-6). However, there have been few comparisons across species(7,8), in stark contrast with genomics initiatives(9).
[more...]
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Accurate annotation of human protein-coding small open reading frames
Nature chemical biology. 2020. Martinez, TF et al. Salk Inst Biol Studies, Clayton Fdn Labs Peptide Biol, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.
ABSTRACT: Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs.
[more...]
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Persulfidation-based modification of cysteine desulfhydrase and the NADPH oxidase RBOHD controls guard cell abscisic acid signaling
The Plant Cell. 2020. Shen, J et al. Nanjing Agr Univ, Coll Life Sci, Lab Ctr Life Sci, Nanjing 210095, Peoples R China.
ABSTRACT: A persulfidation-based reversible post-translational modification of Cys desulfhydrase and NADPH oxidase RBOHD fine-tunes guard cell ABA signaling. Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific Cys residues to form persulfides. However, the mechanisms that underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore, analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana).
[more...]
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Pre-termination transcription complex: structure and function
Molecular cell. 2020. Hao, ZT et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT: Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex'' (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC.
[more...]
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A streamlined mass spectrometry–based proteomics workflow for large‐scale FFPE tissue analysis
The Journal of pathology. 2020. Coscia, F et al. Max Planck Inst Biochem, D-82152 Martinsried, Germany.
ABSTRACT: Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides.
[more...]
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Cancer neoantigen prioritization through sensitive and reliable proteogenomics analysis
Nature communications. 2020. Wen, B et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: Genomics-based neoantigen discovery can be enhanced by proteomic evidence, but there remains a lack of consensus on the performance of different quality control methods for variant peptide identification in proteogenomics. We propose to use the difference between accurately predicted and observed retention times for each peptide as a metric to evaluate different quality control methods. To this end, we develop AutoRT, a deep learning algorithm with high accuracy in retention time prediction. Analysis of three cancer data sets with a total of 287 tumor samples using different quality control strategies results in substantially different numbers of identified variant peptides and putative neoantigens.
[more...]
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Identification of novel DPP–IV inhibitory peptides from Atlantic salmon (Salmo salar) skin
Food Research International. 2020. Jin, RT et al. Northeast Agr Univ, Coll Food Sci, 600 Changjiang Rd, Harbin 150030, Peoples R China.
ABSTRACT: The aim of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from salmon skin collagen hydrolysate, and to evaluate the possible inhibition mechanism of DPP-IV and peptide. Salmon skin collagen was hydrolyzed by pepsin, trypsin, papain, or Alcalase 2.4 L, separately. Trypsin hydrolysate (10 mg/mL) showed the highest inhibitory activity of 66.12 +/- 0.68%. The hydrolysate was separated into three fractions by ultrafiltration, and the inhibitory IC50 of M1 (molecular weight< 3 kDa) was 1.54 +/- 0.06 mg/mL.
[more...]
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Characterization of urinary exosomes purified with size exclusion chromatography and ultracentrifugation
Journal of Proteome Research. 2020. Guan, S et al. Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China.
ABSTRACT: Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years.
[more...]
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TransCirc: an interactive database for translatable circular RNAs based on multi-omics evidence
Nucleic Acids Research. 2020. Huang, WD et al. Chinese Acad Sci, Biomed Big Data Ctr, CAS MPG Partner Inst Computat Biol, CAS Key Lab Computat Biol,Shanghai Inst Nutr & Hl, Shanghai 200031, Peoples R China.
ABSTRACT: TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores froma machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions.
[more...]
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An Arabidopsis secondary metabolite directly targets expression of the bacterial type III secretion system to inhibit bacterial virulence
Cell host & microbe. 2020. Wang, W et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing 100101, Peoples R China.
ABSTRACT: Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P.
[more...]
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A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes
NATURE PROTOCOLS. 2020. Fu, L et al. Beijing Inst Life, State Key Lab Prote, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, Beijing, Peoples R China.
ABSTRACT: Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses.
[more...]
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Structural snapshots of human pre-60S ribosomal particles before and after nuclear export
Nature communications. 2020. Liang, XM et al. Peking Univ, Peking Tsinghua Joint Ctr Life Sci, Sch Life Sci, State Key Lab Membrane Biol, Beijing 100871, Peoples R China.
ABSTRACT: Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk.
[more...]
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Pervasive translation of circular RNAs driven by short IRES-like elements
Nature communications. 2020. Fan, XJ et al. Shanghai Inst Nutr & Hlth, Biomed Big Data Ctr, CAS Ctr Excellence Mol Cell Sci, CAS Key Lab Computat Biol, Shanghai, Peoples R China; Chinese Acad Sci, Univ Chinese Acad Sci, Beijing, Peoples R China
ABSTRACT: Some circular RNAs (circRNAs) were found to be translated through IRES-driven mechanism, however the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we develop a cell-based system to screen random sequences and identify 97 overrepresented hexamers that drive cap-independent circRNA translation. These IRES-like short elements are significantly enriched in endogenous circRNAs and sufficient to drive circRNA translation.
[more...]
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Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation
Elife. 2020. Pan, ZQ et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity.
[more...]
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Investigation of indigoidine synthetase reveals a conserved active-site base residue of nonribosomal peptide synthetase oxidases
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2020. Pang, B et al. Univ Calif Berkeley, QB3 Inst, Berkeley, CA 94720 USA.
ABSTRACT: Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound L-glutaminyl residue.
[more...]
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SlFERL interacts with S-adenosylmethionine synthetase to regulate fruit ripening
Plant Physiology. 2020. Ji, DC et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Bot, Key Lab Plant Resources, Beijing 100093, Peoples R China.
ABSTRACT: The tomato membrane protein SlFERL regulates fruit ripening via modulating ethylene production. Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato (Solanum lycopersicum) FERONIA Like (SlFERL), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses.
[more...]
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Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase
Nature microbiology. 2020. Cox, RM et al. Georgia State Univ, Inst Biomed Sci, Atlanta, GA 30303 USA.
ABSTRACT: Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping.
[more...]
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A clickable APEX probe for proximity-dependent proteomic profiling in yeast
Cell Chemical Biology. 2020. Li, Y et al. Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn,Minist Educ, Beijing Natl Lab Mol Sci,Synthet & Funct Biomol C, Beijing 100871, Peoples R China.
ABSTRACT: The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP.
[more...]
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Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's …
Journal of proteomics. 2020. de Lima, LP et al. Inst Butantan, Lab Especial Ciclo Celular, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, Brazil.
ABSTRACT: Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy.
[more...]
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A comprehensive evaluation of MS/MS spectrum prediction tools for shotgun proteomics
Proteomics. 2020. Xu, R et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Spectrum prediction using machine learning or deep learning models is an emerging method in computational proteomics. Several deep learning-based MS/MS spectrum prediction tools have been developed and showed their potentials not only for increasing the sensitivity and accuracy of data-dependent acquisition search engines, but also for building spectral libraries for data-independent acquisition analysis. Different tools with their unique algorithms and implementations may result in different performances.
[more...]
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Global lysine crotonylation profiling of mouse liver
Proteomics. 2020. Liu, JF et al. Chinese Acad Med Sci & Peking Union Med Coll, Inst Basic Med Sci, Dept Biochem & Mol Biol, State Key Lab Med Mol Biol, Beijing 100005, Peoples R China.
ABSTRACT: Lysine crotonylation (Kcr) is a recently discovered post-translational modification that potentially regulates multiple biological processes. With an objective to expand the available crotonylation datasets, LC-MS/MS is performed using mouse liver samples under normal physiological conditions to obtain in vivo crotonylome. A label-free strategy is used and 10 034 Class I (localization probabilities > 0.75) crotonylated sites are identified in 2245 proteins. The KcrE, KcrD, and EKcr motifs are significantly enriched in the crotonylated peptides.
[more...]
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Synergistic optimization of Liquid Chromatography and Mass Spectrometry parameters on Orbitrap Tribrid mass spectrometer for high efficient data‐dependent …
Journal of Mass Spectrometry. 2020. Huang, PW et al. Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China.
ABSTRACT: Steady improvement in Orbitrap-based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In-depth analysis of the performance of state-of-the-art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data-dependent acquisition approaches, with Orbitrap as the first-stage (MS1) mass analyzer and the same Orbitrap (high-high approach) or ion trap (high-low approach) as the second-stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer.
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Rapid and easy enrichment strategy for naturally acetylated N termini based on LysN digestion and amine-reactive resin capture
Analytical chemistry. 2020. Du, XX et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Protein N-terminal acetylation (N-alpha-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that N-alpha-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of N-alpha-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini.
[more...]
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Unambiguous phosphosite localization through the combination of trypsin and lysarginase mirror spectra in a large-scale phosphoproteome study
Journal of proteome research. 2020. Xu, F et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side.
[more...]
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An Algorithm to Improve the Speed of Semi and Non-Specific Enzyme Searches in Proteomics
Current bioinformatics. 2020. Rolfs, Z et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT: Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time-consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and nonspecific enzyme searches and could be applied to existing search programs. Methods: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once.
[more...]
Use: pFind



Characterization of lysine monomethylome and methyltransferase in model Cyanobacterium Synechocystis Sp. PCC 6803
Genomics, proteomics & bioinformatics. 2020. Lin, XH et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT: Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp.
[more...]
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Open Search Strategy for Inferring the Masses of Cross-Link Adducts on Proteins
Analytical Chemistry. 2020. Slavin, M et al. Hebrew Univ Jerusalem, Inst Life Sci, IL-9190401 Jerusalem, Israel.
ABSTRACT: Development of new reagents for protein cross-linking is constantly ongoing. The chemical formulas for the linker adducts formed by these reagents are usually deduced from expert knowledge and then validated by mass spectrometry. Clearly, it would be more rigorous to infer the chemical compositions of the adducts directly from the data without any prior assumptions on their chemistries. Unfortunately, the analysis tools that are currently available to detect chemical modifications on linear peptides are not applicable to the case of two cross-linked peptides.
[more...]
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Global characterization of modifications to the charge isomers of IgG antibody
Journal of Pharmaceutical Analysis. 2020. Cui, XL et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing
ABSTRACT: Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels.
[more...]
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Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing
JOURNAL OF SEPARATION SCIENCE. 2020. Yang, C et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT: Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS(2)spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase).
[more...]
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Protocol for Proximity-Dependent Proteomic Profiling in Yeast Cells by APEX and Alk-Ph Probe
STAR Protocols. 2020. Li, Yi et al. Peking University
ABSTRACT: Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome.
[more...]
Use: pQuant; pFind



Open-pFind verified four missing proteins from multi-tissues
Journal of proteome research. 2020. Wu, SJ et al. Wuhan Univ, Sch Basic Med Sci, Minist Educ, Key Lab Combinatl Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.
ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on.
[more...]
Use: pFind



A protein identification algorithm optimization for mass spectrometry data using deep learning
2020 3rd International Conference on Advanced Electronic Materials, Computers and Software Engineering (AEMCSE). 2020. Xu, Rui et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center National Center for Protein Sciences (Beijing), Beijing
ABSTRACT: Protein sequence database search is one of the most commonly used methods for protein identification in shotgun proteomics. In tradition, searching a protein sequence database is usually required to construct the theoretical spectrum for each peptide at first, which only considers the information of mass-to-charge ratio at present. However, the information related to isotope peak intensity is neglected. Thanks to the rapid development of artificial intelligence technique in recent years, deep learning-based MS/MS spectrum prediction tools have showed a high accuracy and great potentials to improve the sensitivity and accuracy of protein sequence database searching.
[more...]
Use: pFind; pDeep



健康人的尿液中为什么会有蛋白质?
中国科学. 生命科学. 2020. 刘永涛 et al. 北京医院国家老年医学中心检验医学科, 北京 100730, 中国; 北京师范大学生命科学学院, 生物化学与分子生物学系, 基因工程药物及生物技术北京市重点实验室, 北京 100875, 中国
ABSTRACT: Proteins are essential nutrients for humans, but why is there a small amount of proteins excreted through urine? Although healthy people have low levels of protein in the urine, they are rich in variety.The proteins in them may be unnecessary wastes of the body or may even have a toxic effect on the body and must be excreted.Compared with small-molecule metabolites, proteins have a complex structure and various functions.Even a small change at the molecular level will affect their subsequent biological functions.A comprehensive comparison of the molecular level modifications of plasma and urine proteome, the difference may provide clues to the presence of protein in the urine.In this study, a total of 9 healthy people's urine and 9 healthy people's plasma samples were collected.The samples were analyzed by high-resolution tandem mass spectrometry and unlabeled quantitative proteomics techniques, and the non-limiting modification identification algorithm was used to to compare molecular modifications between two types of samples as a whole.The results showed that the amount of cysteine(Cys)modification to dehydroalanine(Dha)in the urine proteome was higher than that in plasma.The molecular modification[CysDha] destroys the disulfide bond of the original protein,thereby affecting or even changing the structure and biological function of the protein.Therefore, this study revealed the differences in the overall proteome modification of plasma and urine and pointed out that the structure of protein may be irreversibly changed due to the difference in modification of protein molecules, which in turn caused proteins which lost their functions and even toxic proteins from the blood to be excreted into the urine.
Use: pFind



Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle
Journal of proteomics. 2020. de Lima, LP et al. Inst Butantan, Lab Especial Ciclo Celular, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, Brazil.
ABSTRACT: Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy.
[more...]
Use: pFind



Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp. PCC 6803
Genomics, proteomics & bioinformatics. 2020. Lin, XH et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.
ABSTRACT: Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp.
[more...]
Use: pFind



Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study
Journal of proteome research. 2020. Xu, F et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side.
[more...]
Use: pFind



UPEFinder: A Bioinformatic Tool for the Study of Uncharacterized Proteins Based on Gene Expression Correlation and the PageRank Algorithm
Journal of proteome research. 2020. Gonzalez-Gomariz, J et al. Navarra Inst Hlth Res, IdiSNA, E-31008 Pamplona, Spain.
ABSTRACT: The Human Proteome Project (HPP) is leading the international effort to characterize the human proteome. Although the main goal of this project was first focused on the detection of missing proteins, a new challenge arose from the need to assign biological functions to the uncharacterized human proteins and describe their implications in human diseases. Not only the proteins with experimental evidence (uPE1 proteins) but also the uncharacterized missing proteins (uMPs) were the objects of study in this challenge, neXt-CP50.
[more...]
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Quantitative Perspective on Online Flow Reaction Profiling Using a Miniature Mass Spectrometer
ORGANIC PROCESS RESEARCH & DEVELOPMENT. 2020. Sheng, HM et al. Merck & Co Inc, Analyt Sci, Rahway, NJ 07065 USA.
ABSTRACT: Online mass spectrometry has proven to be a useful tool for characterizing many aspects of chemical reactions. However, to the best of the authors' knowledge, no reference standard (RS) quantitation approach has been applied in online MS profiling work to date. In this study, we present a RS approach for online quantitation of an aerobic oxidation reaction in flow using a miniature mass spectrometer, with both internal RS and external RS quantitation approaches being evaluated. Quinoline, a structurally similar and chemically inert compound under these reaction conditions, was chosen as the RS to quantify the pyridine aldehyde product.
[more...]
Use: pFind



Open-pFind Verified Four Missing Proteins from Multi-Tissues
Journal of proteome research. 2020. Wu, SJ et al. Wuhan Univ, Sch Basic Med Sci, Minist Educ, Key Lab Combinatl Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.
ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on.
[more...]
Use: pFind



D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery
Journal of proteome research. 2020. Zhang, YL et al. BGI Shenzhen, BGI Genom, Shenzhen 518083, Guangdong, Peoples R China.
ABSTRACT: Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs.
[more...]
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Synergistic optimization of Liquid Chromatography and Mass Spectrometry parameters on Orbitrap Tribrid mass spectrometer for high efficient data-dependent proteomics
Journal of Mass Spectrometry. 2020. Huang, PW et al. Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China.
ABSTRACT: Steady improvement in Orbitrap-based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In-depth analysis of the performance of state-of-the-art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data-dependent acquisition approaches, with Orbitrap as the first-stage (MS1) mass analyzer and the same Orbitrap (high-high approach) or ion trap (high-low approach) as the second-stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer.
[more...]
Use: pFind; pParse



Activation of the CARD8 inflammasome requires a disordered region
Cell reports. 2020. Chui, AJ et al. Mem Sloan Kettering Canc Ctr, Triinst PhD Program Chem Biol, New York, NY 10065 USA.
ABSTRACT: Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments.
[more...]
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Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle
Food Chemistry. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT: Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides.
[more...]
Use: pNovo; pFind



Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues
NATURE PROTOCOLS. 2020. Li, QY et al. Univ Calif Davis, Dept Chem, Davis, CA 95616 USA.
ABSTRACT: The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction.
[more...]
Use: pFind; pGlyco



鸽毛滴虫外泌体的分离鉴定及蛋白质谱分析
Acta Veterinaria et Zootechnica Sinica (畜牧兽医学报). 2020. Ni, xinai et al. 中国农业科学院北京畜牧兽医研究所
ABSTRACT: The aim of this study was to determine whether Trichomonas gallinae(T.gallinae)secrete exosomes and investigate the protein composition of exosomes derived fromT.gallinae.T.gallinae was isolated from upper digestive tract of infected pigeons and identified by microscopy and sequencing.Exosomes were extracted from serum absent culture medium,and further verified by transmission electron microscopy,Western blot and nanoparticle tracking analysis(NTA).Label-free was used to identify the proteins of exosomes.Results showed that the parasite had T.gallinae morphology,and sequence alignment of ITS1/5.8S/ITS2gene showed an identity of 98%.The isolated vesicles presented a typical cup-shaped morphology;NTA results showed that particle size concentrated on 125.1nm,percentage of peak diameter at 132.3nm was 99.3%;proteins such as CD63and TSG101were conspicuously detected,suggesting that the extracts were exosomes.Mass spectrometry results showed that enolase,glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate mutase and elongation factor were the highly expressed proteins.GO pathways showed that proteins of exosomes mainly enriched into cellular components such as intracellular and cytoplasmic,played GTP binding and GTPase activity function, and participated in biological process of small GTPase mediated signal transduction and glycolytic process.KEGG enrichment analysis showed that proteins of exosomes were enriched in pathways as metabolic pathways,glycolysis/gluconeogenesis,biosynthesis of antibiotics and secondary metabolites.These results indicated that T.gallinaecan secrete exosomes and proteins of exosomes might play roles in energy metabolism,signal transduction and biosynthesis.
Use: pFind



Proteomic Analysis Reveals that EPHX1 Contributes to 5-Fluorouracil Resistance in a Human Hepatocellular Carcinoma Cell Line
Proteomics Clinical Applications. 2020. Sun, R et al. Dalian Med Univ, Dept Gen Surg, Div Hepatobiliary & Pancreat Surg, Affiliated Hosp 2, Dalian 116023, Peoples R China.
ABSTRACT: Purpose The extensive drug resistance of hepatocellular carcinoma (HCC) has become a major cause of chemotherapy failure. A deeper understanding of the drug resistance mechanism of tumor cells is very significant for improving the clinical prognosis of patients with HCC. Experimental Design In this study, proteomic studies on the composition of 5-fluorouracil (5-Fu) resistant Bel/5Fu cell line and its parent Bel7402 cell line by using an ionic liquid assisted proteins extraction method with the advantage of extracting plasma membrane proteins to a wider extent are performed.
[more...]
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Sequence analysis of digestion-resistant peptides may be an efficient strategy for studying the linear epitopes of Jug r 1, the major walnut allergen
Food Chemistry. 2020. Guo, XY et al. China Agr Univ, Coll Food Sci & Nutr Engn, Beijing Adv Innovat Ctr Food Nutr & Human Hlth, 17 Qinghua East Rd, Beijing, Peoples R China.
ABSTRACT: Jug r 1, the major allergen of walnut, triggers severe allergic reactions through epitopes. Hence, research on the efficient strategy for analyzing the linear epitopes of Jug r 1 are necessary. In this work, bioinformatics analysis was used to predict the linear epitopes of Jug r 1. Overlapping peptide synthesis was used to map linear epitopes. In vitro simulated gastrointestinal digestion and HPLC-MS/MS were used to identify digestion-resistant peptides. The results showed that six predicted linear epitopes were AA28-35, AA42-49, AA55-62, AA65-73, AA97-104, and AA109-121.
[more...]
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Parallel channels-multidimensional protein identification technology
Journal of the American Society for Mass Spectrometry. 2020. Zhang, N et al. Peking Univ, Sch Pharmaceut Sci, Sch Basic Med Sci, Hlth Sci Ctr,Ctr Precis Med Multi Res, Beijing 100191, Peoples R China.
ABSTRACT: Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology.
[more...]
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Sequential amidation of peptide C-termini for improving fragmentation efficiency
Journal of Mass Spectrometry. 2020. Wu, Q et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Owing to the poor fragmentation efficiency caused by the lack of a positively charged basic group at the C-termini of peptides, the identification of nontryptic peptides in classical proteomics is known to be less efficient. Particularly, attaching positively charged basic groups to C-termini via chemical derivatizations is known to be able to enhance their fragmentation efficiency. In this study, we introduced a novel strategy, C-termini sequential amidation reaction (CSAR), to improve peptide fragmentation efficiency.
[more...]
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Openpepxl: an open-source tool for sensitive identification of cross-linked peptides in XL-MS
Molecular & Cellular Proteomics. 2020. Netz, E et al. Max Planck Inst Dev Biol, Biomol Interact, Tubingen, Germany.
ABSTRACT: XL-MS has been recognized as an effective source of information about protein structures and interactions. OpenPepXL is a sensitive XL-MS identification software that reports from 7% to 40% more structurally validated cross-links than other tools on data sets with available high-resolution structures for cross-link validation. It is open source and has been built as part of the OpenMS suite of tools. OpenPepXL supports all common operating systems and open data formats. Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions.
[more...]
Use: pLink



Vaccinia virus immunomodulator A46: destructive interactions with MAL and MyD88 shown by negative-stain electron microscopy
Structure. 2020. Azar, DF et al. Med Univ Vienna, Vienna Bioctr, Max Perutz Labs, Dr Bohr Gasse 9-3, A-1030 Vienna, Austria.
ABSTRACT: Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88.
[more...]
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Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter
Frontiers in plant science. 2020. Wei, B et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium.
ABSTRACT: In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond.
[more...]
Use: pLink



Epitope and paratope mapping of PD-1/Nivolumab by mass spectrometry-based Hydrogen--deuterium exchange, cross-linking, and molecular docking
Analytical Chemistry. 2020. Zhang, MM et al. Bristol Myers Squibb Co, Nonclin Res & Dev, Pharmaceut Candidate Optimizat, Princeton, NJ 08540 USA.
ABSTRACT: Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen -deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking.
[more...]
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Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation
Elife. 2020. Lv, L et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK).
[more...]
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Rett syndrome-causing mutations compromise MeCP2-mediated liquid–liquid phase separation of chromatin
Cell Research. 2020. Wang, L et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Univ Peking Univ Joint Ctr Life Sci, Sch Life Sci,Beijing Frontier Res Ctr Biol Struct, Beijing, Peoples R China.
ABSTRACT: Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2.
[more...]
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Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state
PLOS Biology. 2020. Bu, WT et al. Nanyang Technol Univ, Sch Biol Sci, Singapore, Singapore.
ABSTRACT: Kindlin-1, -2, and -3 directly bind integrin beta cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin beta cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation.
[more...]
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Expanding the depth and sensitivity of cross-link identification by differential ion mobility using high-field asymmetric waveform ion mobility spectrometry
Analytical Chemistry. 2020. Schnirch, L et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Chem Biol, D-13125 Berlin, Germany.
ABSTRACT: In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker.
[more...]
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Structure of the human sodium leak channel NALCN in complex with FAM155A
Nature communications. 2020. Xie, JF et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China.
ABSTRACT: NALCN, a sodium leak channel expressed mainly in the central nervous system, is responsible for the resting Na+ permeability that controls neuronal excitability. Dysfunctions of the NALCN channelosome, NALCN with several auxiliary subunits, are associated with a variety of human diseases. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 angstroms. FAM155A forms extensive interactions with the extracellular loops of NALCN that may help stabilize NALCN in the membrane.
[more...]
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Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter
Frontiers in plant science. 2020. Wei, B et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium.
ABSTRACT: In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond.
[more...]
Use: pLink



Generation of aggregates of α-lactalbumin by UV-B light exposure
Journal of Agricultural and Food Chemistry. 2020. Zhao, ZC et al. Univ Copenhagen, Fac Sci, Dept Food Sci, DK-1958 Frederiksberg C, Denmark.
ABSTRACT: Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of alpha-lactalbumin (alpha-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by alpha-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation.
[more...]
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Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis
Scientific Reports. 2020. Bruckmann, C et al. IFOM Fdn FIRC, Inst Mol Oncol, Via Adamello 16, I-20139 Milan, Italy.
ABSTRACT: Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1.
[more...]
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Defining the architecture of the human TIM22 complex by chemical crosslinking
FEBS Letters. 2020. Valpadashi, A et al. Univ Med Ctr Gottingen, Dept Cellular Biochem, Humboldtallee 23, D-37073 Gottingen, Germany.
ABSTRACT: The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex.
[more...]
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Molecular topology of RNA polymerase I upstream activation factor
Molecular and Cellular Biology. 2020. Knutson, BA et al. SUNY Upstate Med Univ, Dept Biochem & Mol Biol, Syracuse, NY 13210 USA.
ABSTRACT: Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation.
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OpenPepXL: An open-source tool for sensitive identification of cross-linked peptides in XL-MS
Molecular & Cellular Proteomics. 2020. Netz, E et al. Max Planck Inst Dev Biol, Biomol Interact, Tubingen, Germany.
ABSTRACT: XL-MS has been recognized as an effective source of information about protein structures and interactions. OpenPepXL is a sensitive XL-MS identification software that reports from 7% to 40% more structurally validated cross-links than other tools on data sets with available high-resolution structures for cross-link validation. It is open source and has been built as part of the OpenMS suite of tools. OpenPepXL supports all common operating systems and open data formats. Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions.
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Vaccinia virus immunomodulator A46: destructive interactions with MAL and MyD88 Shown by negative-stain electron microscopy
Structure. 2020. Azar, DF et al. Med Univ Vienna, Vienna Bioctr, Max Perutz Labs, Dr Bohr Gasse 9-3, A-1030 Vienna, Austria.
ABSTRACT: Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88.
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Evidence of allosteric coupling between substrate binding and Adx recognition in the vitamin D carbon-24 hydroxylase CYP24A1
Biochemistry. 2020. Kumar, A et al. Univ Buffalo, Jacobs Sch Med, Dept Biochem, Buffalo, NY 14203 USA.
ABSTRACT: Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1Adx complex.
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DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1
Journal of Molecular Biology. 2020. Kang, JJ et al. Inst Rech Clin Montreal, 110 Ave Pins Ouest, Montreal, PQ H2W 1R7, Canada.
ABSTRACT: Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro.
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The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering
Journal of cell science. 2020. Kukkurainen, S et al. Tampere Univ, Fac Med & Hlth Technol, Arvo Ylpon Katu 34, FI-33520 Tampere, Finland.
ABSTRACT: Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the beta integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate beta 3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain.
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FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho
PNAS. 2020. Suzuki, Y et al. Yale Univ, Dept Pharmacol, Sch Med, New Haven, CT 06510 USA.
ABSTRACT: The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of alpha-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA.
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Integrated structural modeling of full-length LRH-1 reveals inter-domain interactions contribute to receptor structure and function
Structure. 2020. Seacrist, CD et al. Vanderbilt Univ, Dept Pharmacol, Nashville, TN 37235 USA.
ABSTRACT: Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS, The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2, We confirmed several other predicted inter-domain interactions via structural and functional analyses.
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Dynamic folding modulation generates FGF21 variant against diabetes
EMBO reports. 2020. Zhu, Lei et al. High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy ofSciences, Hefei, China
ABSTRACT: Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible beta-trefoil conformation affects the folding of beta2-beta3 hairpin and further overall protein stability.
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A structural model of the endogenous human BAF complex informs disease mechanisms
Cell. 2020. Mashtalir, N et al. Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02115 USA.
ABSTRACT: Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal alpha-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities.
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Structure of the transcription coactivator SAGA
Nature. 2020. Haibo Wang et al. Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany
ABSTRACT: Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)(1,2) and transcription factor IID (TFIID)(2-4). SAGA is required for all regulated transcription(5) and is conserved among eukaryotes(6). SAGA contains four modules(7-9): the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures(10-14), but the structure of the central core module is unknown.
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Structure of complete Pol II–DSIF–PAF–SPT6 transcription complex reveals RTF1 allosteric activation
Nature Structural & Molecular Biology. 2020. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four alpha-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel.
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Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome
Nature. 2020. Wagner, FR et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: The cryo-electron microscopy structure of the 16-subunit yeast SWI/SNF complex RSC in complex with a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes. Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)(1,2). In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1(4,5).
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Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation
Science (New York, N.Y.). 2020. Townsend, Cole et al. Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
ABSTRACT: Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter.
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Molecular architecture of the human 17S U2 snRNP
Nature. 2020. Zhang, ZW et al. MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.
ABSTRACT: The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing'. Stable addition of U2 during early spliceosome formation requiresthe DEAD-box ATPase PRP5(2-7). Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)(8), but whether the human U2 snRNA folds in a similar manner is unknown.
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A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes
Nature Communications. 2020. Beveridge, R et al. Vienna Bioctr VBC, Res Inst Mol Pathol IMP, Campus Vienna Bioctr 1, A-1030 Vienna, Austria.
ABSTRACT: Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%.
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A basic motif anchoring ISWI to nucleosome acidic patch regulates nucleosome spacing
Nature chemical biology. 2020. Dao, HT et al. Princeton Univ, Dept Chem, Princeton, NJ USA.
ABSTRACT: A photocrosslinking-based nucleosome profiling approach is used to identify a conserved basic motif in the ISWI remodeler SNF2h that anchors it to the acidic patch of nucleosome and enables nucleosome sliding activity. Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We used a photocrosslinking-based nucleosome profiling technology (photoscanning) to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope.
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A molecular switch regulating transcriptional repression and activation of PPARγ
Nature communications. 2020. Shang, JS et al. Scripps Res Inst, Dept Integrat Struct & Computat Biol, Jupiter, FL 33458 USA.
ABSTRACT: Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume.
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High-density chemical cross-linking for modeling protein interactions
PNAS. 2020. Mintseris, J et al. Harvard Med Sch, Dept Cell Biol, Boston, MA 02115 USA.
ABSTRACT: Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate.
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A cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria
Molecular & Cellular Proteomics. 2020. Linden, A et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, Gottingen, Germany.
ABSTRACT: Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system.
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Epitope and Paratope Mapping of PD-1/Nivolumab by Mass Spectrometry-Based Hydrogen–Deuterium Exchange, Cross-linking, and Molecular Docking
Analytical Chemistry. 2020. Zhang, MM et al. Bristol Myers Squibb Co, Nonclin Res & Dev, Pharmaceut Candidate Optimizat, Princeton, NJ 08540 USA.
ABSTRACT: Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen -deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking.
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Discovery of a regulatory subunit of the yeast fatty acid synthase
Cell. 2020. Singh, K et al. Max Planck Inst Biophys Chem, Dept Struct Dynam, D-37077 Gottingen, Germany.
ABSTRACT: Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a gamma subunit of the 2.6 megadalton alpha(6)-beta(6) S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions.
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Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs
Scientific Reports. 2020. Kaziales, A et al. Tech Univ Munich, Dept Chem, Ctr Integrated Prot Sci Munich, Lichtenbergstr 4, D-85748 Garching, Germany.
ABSTRACT: The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies.
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Structural insights into the roles of metazoan-specific splicing factors in the human step 1 spliceosome
Molecular Cell. 2020. Bertram, K et al. MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 angstrom core resolution and 4.5-5.7 angstrom at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation.
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The nucleosome remodeling and deacetylase complex has an asymmetric, dynamic, and modular architecture
Cell reports. 2020. Low, JKK et al. Univ Sydney, Sch Life & Environm Sci, Sydney, NSW, Australia.
ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics.
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A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies
Nature Communications. 2020. Macleod, OJS et al. Univ Cambridge, Dept Biochem, Tennis Court Rd, Cambridge CB2 1QW, England.
ABSTRACT: Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface.
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Functional and mass spectrometric evaluation of an anti-tick antigen based on the P0 peptide conjugated to Bm86 protein
Pathogens. 2020. Mallon, AR et al. Ctr Genet Engn & Biotechnol CIGB, Anim Biotechnol Dept, Havana 10600, Cuba.
ABSTRACT: A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin fromMegathura crenulataand used as an immunogen againstRhipicephalus microplusandRhipicephalus sanguineuss.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate againstAmblyomma mixtumandIxodes ricinusspecies, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein.
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Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau
eLife. 2020. Ma, XJ et al. Chinese Acad Sci, Interdisciplinary Res Ctr Biol & Chem, Shanghai Inst Organ Chem, Shanghai, Peoples R China.
ABSTRACT: Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD(+) synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model.
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Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats
PLOS ONE. 2020. Sajko, S et al. Univ Vienna, Dept Struct & Computat Biol, Max Perutz Labs, Vienna, Austria.
ABSTRACT: MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1.
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Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S
Science Signaling. 2020. Liess, AKL et al. Univ Wurzburg, Rudolf Virchow Ctr Integrat & Translat Bioimaging, D-97080 Wurzburg, Germany.
ABSTRACT: At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover.
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Switch from polymorphic to homogenous self‐assembly of virus‐like particles of simian virus 40 through double‐cysteine substitution
Small. 2020. Xu, CC et al. Guangzhou Med Univ, Guangzhou Women & Childrens Med Ctr, Guangzhou 510623, Peoples R China.
ABSTRACT: Self-assembled virus-like particles (VLPs) hold great potential as natural nanomaterials for applications in many fields. For such purposes, monodisperse size distribution is a desirable property. However, the VLPs of simian virus 40 (SV40), a representative VLP platform, are characterized by polymorphism. In an attempt to eliminate the polymorphism, 15 mutants of the VLP subunit (VP1) are constructed through the substitution of double cysteines at the VP1 pentamer interfaces, generating a group of VLPs with altered size distributions.
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Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade
Journal of Biological Chemistry. 2020. Tossounian, MA et al. VUB, VIB, Ctr Structural Biol, B-1050 Brussels, Belgium.
ABSTRACT: Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms.
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Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms
Journal of Proteome Research. 2020. Zhao, BQ et al. Indiana Univ, Dept Chem, Bloomington, IN 47405 USA.
ABSTRACT: Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS3) are analyzed. Two approaches are introduced to combine interpretations from the above four methods.
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Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails
JOURNAL OF STRUCTURAL BIOLOGY. 2020. Giglio, ML et al. Consejo Nacl Invest Cient & Tecn, Fdn Inst Leloir, IIBBA, Av Patricias Argentinas 435,C1405BWE, Buenos Aires, DF, Argentina.
ABSTRACT: The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 angstrom resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS).
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Decision tree searching strategy to boost the identification of cross-linked peptides
Analytical Chemistry. 2020. Huang, R et al. ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China.
ABSTRACT: We describe an efficient decision tree searching strategy (DTSS) to boost the identification of cross-linked peptides. The DTSS approach allows the identification of a wealth of complementary information to facilitate the construction of more protein-protein interaction networks for human cell lysate, which was tested by the use of a recently reported cross-linking data set (ACS Cent. Sci. 2019, 5, 1514-1522). A variant of the PhoX-linker, named pDSPE, was synthesized and applied to cross-link Escherichia coli cell lysate to demonstrate that the acquisition of doubly charged ions can significantly improve identification results.
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An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples
Journal of proteomics. 2020. Parfentev, I et al. Max Planck Inst Biophys Chem, Res Grp Bioanalyt Mass Spectrometry, Gottingen, Germany.
ABSTRACT: Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n 2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions.
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crisscrosslinkeR: identification and visualization of protein–RNA and protein–protein interactions from crosslinking mass spectrometry
Bioinformatics. 2020. Gail, EH et al. Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic 3800, Australia.
ABSTRACT: The Summary: Unbiased detection of protein-protein and protein-RNA interactions within ribonucleoprotein complexes are enabled through crosslinking followed by mass spectrometry. Yet, different methods detect different types of molecular interactions and therefore require the usage of different software packages with limited compatibility. We present crisscrosslinkeR, an R package that maps both protein-protein and protein-RNA interactions detected by different types of approaches for crosslinking with mass spectrometry.
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ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity
Redox biology. 2020. Costa, RAPE et al. CNPEM, Lab Nacl Biociencias, LNBio, Campinas, SP, Brazil.
ABSTRACT: The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1.
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Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter
Frontiers in plant science. 2020. Wei, B et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium.
ABSTRACT: In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond.
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Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry
Analytical Chemistry. 2020. Schnirch, L et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Chem Biol, D-13125 Berlin, Germany.
ABSTRACT: In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker.
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Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain Electron Microscopy
Structure. 2020. Azar, DF et al. Med Univ Vienna, Vienna Bioctr, Max Perutz Labs, Dr Bohr Gasse 9-3, A-1030 Vienna, Austria.
ABSTRACT: Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88.
[more...]
Use: pLink



FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with $\alpha$-Klotho
PNAS. 2020. Suzuki, Y et al. Yale Univ, Dept Pharmacol, Sch Med, New Haven, CT 06510 USA.
ABSTRACT: The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of alpha-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA.
[more...]
Use: pLink



Switch from Polymorphic to Homogenous Self-Assembly of Virus-Like Particles of Simian Virus 40 through Double-Cysteine Substitution
Small. 2020. Xu, CC et al. Guangzhou Med Univ, Guangzhou Women & Childrens Med Ctr, Guangzhou 510623, Peoples R China.
ABSTRACT: Self-assembled virus-like particles (VLPs) hold great potential as natural nanomaterials for applications in many fields. For such purposes, monodisperse size distribution is a desirable property. However, the VLPs of simian virus 40 (SV40), a representative VLP platform, are characterized by polymorphism. In an attempt to eliminate the polymorphism, 15 mutants of the VLP subunit (VP1) are constructed through the substitution of double cysteines at the VP1 pentamer interfaces, generating a group of VLPs with altered size distributions.
[more...]
Use: pLink



Generation of aggregates of $\alpha$-lactalbumin by UV-B light exposure
Journal of Agricultural and Food Chemistry. 2020. Zhao, ZC et al. Univ Copenhagen, Fac Sci, Dept Food Sci, DK-1958 Frederiksberg C, Denmark.
ABSTRACT: Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of alpha-lactalbumin (alpha-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by alpha-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation.
[more...]
Use: pLink



Structure of complete Pol II--DSIF--PAF--SPT6 transcription complex reveals RTF1 allosteric activation
Nature Structural & Molecular Biology. 2020. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four alpha-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel.
[more...]
Use: pLink



The acetyltransferase Eco1 elicits cohesin dimerization during S phase
Journal of Biological Chemistry. 2020. Shi, D et al. China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100193, Peoples R China.
ABSTRACT: Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast (Saccharomyces cerevisiae) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial.
[more...]
Use: pLink



Integrative structure and function of the yeast exocyst complex
Protein Science. 2020. Ganesan, SJ et al. Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, Byers Hall,1700 4th St,Suite 503B, San Francisco, CA 94158 USA.
ABSTRACT: Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level.
[more...]
Use: pXtract; pLink



Fragment mass spectrum prediction facilitates site localization of phosphorylation
Journal of Proteome Research. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.
ABSTRACT: Liquid chromatography tandem mass spectrometry (LCMS/MS) has been the most widely used technology for phosphoproteomics studies. As an alternative to database searching and probability-based phosphorylation site localization approaches, spectral library searching has been proved to be effective in the identification of phosphopeptides. However, incompletion of experimental spectral libraries limits the identification capability. Herein, we utilize MS/MS spectrum prediction coupled with spectral matching for site localization of phosphopeptides.
[more...]
Use: pDeep; pNovo



Identification and dereplication of endophytic Colletotrichum strains by MALDI TOF mass spectrometry and molecular networking
Scientific reports. 2020. Barthelemy, M et al. Univ Paris Saclay, Inst Chim Subst Nat, CNRS, UPR 2301, Ave Terrasse, F-91198 Gif Sur Yvette, France.
ABSTRACT: The chemical diversity of biologically active fungal strains from 42 Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software.
[more...]
Use: pNovo



Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula × Tina
Journal of experimental botany. 2020. Kocab, O et al. Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
ABSTRACT: Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion.
[more...]
Use: pNovo



Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula$\times$ Tina
Journal of experimental botany. 2020. Kocab, O et al. Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic
ABSTRACT: Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion.
[more...]
Use: pNovo



Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics
Analytical Chemistry. 2020. Capriotti, AL et al. Sapienza Univ Roma, Dept Chem, I-00185 Rome, Italy.
ABSTRACT: Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+.
[more...]
Use: pNovo



A new opening for the tricky untargeted investigation of natural and modified short peptides
Talanta. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.
ABSTRACT: Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose.
[more...]
Use: pNovo



Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.
FASEB JOURNAL. 2020. Biggart, MGS et al. St George’s University Of London
ABSTRACT: The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides.
[more...]
Use: pNovo



A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics
Nature Communications. 2020. Fang, P et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.
ABSTRACT: Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples.
[more...]
Use: pGlyco; pParse



Identifying sialylation linkages at the glycopeptide level by glycosyltransferase labeling assisted mass spectrometry (GLAMS)
Analytical chemistry. 2020. Zhu, H et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of alpha 2,3/alpha 2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy.
[more...]
Use: pParse; pGlyco



基于多头注意力机制和残差神经网络的肽谱匹配打分算法
Journal of Computer Applications. 2020. 闵鑫 et al. 山东理工大学计算机科学与技术学院
ABSTRACT: Peptide spectrum match scoring algorithm plays a key role in the peptide sequence identification,and the traditional scoring algorithm cannot effectively make full use of the peptide fragmentation pattern to perform scoring. In order to solve the problem,a multi-classification probability sum scoring algorithm combined with the peptide sequence information representation called deepscore- alpha was proposed. In this algorithm,the second scoring was not performed with the consideration of global information,and there was no limitation on the similarity calculation method of theoretical mass spectrum and experimental mass spectrum.
[more...]
Use: pParse; pDeep



Computational classification of core and outer fucosylation of N-glycoproteins in human plasma using collision-induced dissociation in mass spectrometry
Rapid Communications in Mass Spectrometry. 2020. Jeong, HK et al. Korea Basic Sci Inst, Res Ctr Bioconvergence Anal, 162 Yeongudanji Ro, Cheongju 28119, South Korea.
ABSTRACT: Rationale Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods The relative intensities of the Y1F, Y2F, Y3F, and Y4F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation.
[more...]
Use: pGlyco



Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor
Cell host & microbe. 2020. Zhao, P et al. Univ Georgia, Dept Biochem & Mol Biol, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA.
ABSTRACT: The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another.
[more...]
Use: pGlyco



Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications
Engineering (Beijing, China). 2020. Sun, Zeyu et al. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310011, China
ABSTRACT: The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2.
[more...]
Use: pGlyco



Relative retention time estimation improves N-glycopeptide identifications by LC--MS/MS
Journal of proteome research. 2020. Klein, J et al. Boston Univ, Program Bioinformat, Boston, MA 02215 USA.
ABSTRACT: Glycopeptides identified by tandem mass spectrometry rely on the identification of the peptide backbone sequence and the attached glycan(s) by the incomplete fragmentation of both moieties. This may lead to ambiguous identifications where multiple structures could explain the same spectrum equally well due to missing information in the mass spectrum or incorrect precursor mass determination. To date, approaches to solving these problems have been limited, and few inroads have been made to address these issues.
[more...]
Use: pGlyco



Quantitative analysis of $\alpha$-1-antitrypsin glycosylation isoforms in HCC patients using LC-HCD-PRM-MS
Analytical Chemistry. 2020. Yin, HD et al. Hong Kong Polytech Univ, Shenzhen Res Inst, Shenzhen, Peoples R China.
ABSTRACT: The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum alpha-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS.
[more...]
Use: pGlyco



Peptide release after simulated infant in vitro digestion of dry heated cow’s milk protein and transport of potentially immunoreactive peptides across the Caco-2 cell monolayer
Nutrients. 2020. Zenker, HE et al. Wageningen Univ & Res Ctr, Food Qual & Design Grp, NL-6708 WG Wageningen, Netherlands.
ABSTRACT: Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 degrees C) and high temperature (130 degrees C) and applied to simulated infant in vitro digestion.
[more...]
Use: pGlyco



Glycomics-informed glycoproteomic analysis of site-specific glycosylation for SARS-CoV-2 spike protein
STAR protocols. 2020. Rosenbalm, Katelyn E et al. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA
ABSTRACT: This protocol describes an integrated approach for analyzing site-specific N- and O-linked glycosylation of SARS-CoV-2 spike protein by mass spectrometry. Glycoproteomics analyzes intact glycopeptides to examine site-specific microheterogeneity of glycoproteins. Glycomics provides structural characterization on any glycan assignments by glycoproteomics. This procedure can be modified and applied to a variety of N- and/or O-linked glycoproteins. Combined with bioinformatics, the glycomics-informed glycoproteomics may be useful in generating 3D molecular dynamics simulations of certain glycoproteins alone or interacting with one another.
[more...]
Use: pGlyco



Glycoproteomic analysis of human urinary exosomes
Analytical Chemistry. 2020. Brown, CJ et al. Indiana Univ, Dept Chem, Bloomington, IN 47401 USA.
ABSTRACT: Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation.
[more...]
Use: pGlyco



SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides
Bioinformatics. 2020. Schulze, S et al. Univ Penn, Dept Biol, Leidy Labs, Philadelphia, PA 19104 USA.
ABSTRACT: Motivation: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii.
[more...]
Use: pGlyco



An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose
Analytica Chimica Acta. 2020. Wu, MX et al. Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China.
ABSTRACT: A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast.
[more...]
Use: pGlyco



One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides
Analytica Chimica Acta. 2020. Zhang, N et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment.
[more...]
Use: pGlyco



Characterization of N-linked intact glycopeptide signatures of plasma IgGs from patients with prostate carcinoma and benign prostatic hyperplasia for diagnosis pre-stratification
Analyst. 2020. Zhang, Y et al. Sichuan Univ, West China Hosp, MOH, Key Lab Transplant Engn & Immunol, Chengdu 610041, Peoples R China.
ABSTRACT: The discovery of novel non-invasive biomarkers for discriminating between prostate carcinoma (PCa) patients and benign prostatic hyperplasia (BPH) patients is necessary to reduce the burden of biopsies, avoid overdiagnosis and improve quality of life. Previous studies suggest that abnormal glycosylation of immunoglobulin gamma molecules (IgGs) is strongly associated with immunological diseases and prostate diseases. Hence, characterizing N-linked intact glycopeptides of IgGs that correspond to theN-glycan structure with specific site information might enable a better understanding of the molecular pathogenesis and discovery of novel signatures in preoperative discrimination of BPH from PCa.
[more...]
Use: pGlyco



N-glycan structures of target cancer biomarker characterized by two-dimensional gel electrophoresis and mass spectrometry
Analytica Chimica Acta. 2020. Liu, S et al. Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Joint Int Res Lab Metab & Dev Sci, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China.
ABSTRACT: Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry.
[more...]
Use: pGlyco



Quantitative analysis of global protein stability rates in tissues
Scientific reports. 2020. McClatchy, DB et al. Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT: Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases. We present a new technique, QUAD (Quantification of Azidohomoalanine Degradation), to analyze the global degradation rates in tissues using a non-canonical amino acid and mass spectrometry. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability.
[more...]
Use: pQuant



Temporal quantitative profiling of newly synthesized proteins during A$\beta$ accumulation
Journal of Proteome Research. 2020. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT: Accumulation of aggregated amyloid beta (A beta) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of A beta deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice.
[more...]
Use: pQuant



Temporal Quantitative Profiling of Newly Synthesized Proteins during A$\beta$ Accumulation
Journal of Proteome Research. 2020. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT: Accumulation of aggregated amyloid beta (A beta) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of A beta deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice.
[more...]
Use: pQuant



MASH explorer: a universal software environment for top-down proteomics
Journal of proteome research. 2020. Wu, Zhijie et al. Univ Wisconsin, Dept Biostat & Med Informat, Madison, WI 53705 USA; Univ Wisconsin, Carbone Canc Ctr, Madison, WI 53705 USA; Univ Wisconsin, Sch Med & Publ Hlth, Dept Cell & Regenerat Biol, Dept Chem, Madison, WI 53705 USA; Univ Wisconsin, Sch Med & Publ Hlth, Human Prote Program, Madison, WI 53705 USA
ABSTRACT: Top-down mass spectrometry (MS)-based proteomics enable a comprehensive analysis of proteoforms with molecular specificity to achieve a proteome-wide understanding of protein functions. However, the lack of a universal software for top-down proteomics is becoming increasingly recognized as a major barrier, especially for newcomers. Here, we have developed MASH Explorer, a universal, comprehensive, and user-friendly software environment for top-down proteomics. MASH Explorer integrates multiple spectral deconvolution and database search algorithms into a single, universal platform which can process top-down proteomics data from various vendor formats, for the first time.
[more...]
Use: pTop



In silico spectral libraries by deep learning facilitate data-independent acquisition proteomics
Nature communications. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai Stomatol Hosp, Shanghai 200000, Peoples R China.
ABSTRACT: Data-independent acquisition (DIA) is an emerging technology for quantitative proteomic analysis of large cohorts of samples. However, sample-specific spectral libraries built by data-dependent acquisition (DDA) experiments are required prior to DIA analysis, which is time-consuming and limits the identification/quantification by DIA to the peptides identified by DDA. Herein, we propose DeepDIA, a deep learning-based approach to generate in silico spectral libraries for DIA analysis. We demonstrate that the quality of in silico libraries predicted by instrument-specific models using DeepDIA is comparable to that of experimental libraries, and outperforms libraries generated by global models.
[more...]
Use: pDeep



DeepRescore: leveraging deep learning to improve peptide identification in immunopeptidomics
Proteomics. 2020. Li, K et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.
ABSTRACT: The identification of major histocompatibility complex (MHC)-binding peptides in mass spectrometry (MS)-based immunopeptideomics relies largely on database search engines developed for proteomics data analysis. However, because immunopeptidomics experiments do not involve enzymatic digestion at specific residues, an inflated search space leads to a high false positive rate and low sensitivity in peptide identification. In order to improve the sensitivity and reliability of peptide identification, a post-processing tool named DeepRescore is developed.
[more...]
Use: pDeep



Full-spectrum prediction of peptides tandem mass spectra using deep neural network
Analytical chemistry. 2020. Liu, KY et al. Indiana Univ, Sch Informat Comp & Engn, Bloomington, IN 47405 USA.
ABSTRACT: The ability to predict tandem mass (MS/MS) spectra from peptide sequences can significantly enhance our understanding of the peptide fragmentation process and could improve peptide identification in proteomics. However, current approaches for predicting high-energy collisional dissociation (HCD) spectra are limited to predict the intensities of expected ion types, that is, the a/b/c/x/y/z ions and their neutral loss derivatives (referred to as backbone ions). In practice, backbone ions only account for <70% of total ion intensities in HCD spectra, indicating many intense ions are ignored by current predictors.
[more...]
Use: pDeep



Hybrid spectral library combining DIA-MS data and a targeted virtual library substantially deepens the proteome coverage
IScience. 2020. Lou, RH et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China.
ABSTRACT: Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning.
[more...]
Use: pDeep



Structure of nucleosome-bound human BAF complex
SCIENCE. 2020. He, S et al. Fudan
ABSTRACT: Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications.
[more...]
Use: pLink




2019




A protein identification algorithm for tandem mass spectrometry by incorporating the abundance of mRNA into a binomial probability scoring model
Journal of Proteomics. 2019. Ma, WT et al. Hainan Univ, Inst Trop Agr & Forestry, Haikou 570228, Hainan, Peoples R China.
ABSTRACT: Peptide-spectrum matches (PSM) scoring between the experimental and theoretical spectrum is a key step in the identification of proteins using mass spectrometry (MS)-based proteomics analyses. Efficient protein identification using MS/MS data remains a challenge. The strategy of using RNA-seq data increases the number of proteins identified by re-constructing the custom search database and integrating mRNA abundance into the false discovery rate of post-PSM. However, this process lacks an algorithm that can allow the incorporation of mRNA abundance into the key scoring model of PSM.
[more...]
Use: pFind



GenTree, an integrated resource for analyzing the evolution and function of primate-specific coding genes
Genome research. 2019. Shao, Yi et al. Chinese Acad Sci, State Key Lab Integrated Management Pest Insects, Inst Zool, Beijing 100101, Peoples R China; Chinese Acad Sci, CAS Ctr Excellence Anim Evolut & Genet, Kunming 650223, Yunnan, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China; Chinese Acad Sci, Inst Zool, Key Lab Zool Systemat & Evolut, Beijing 100101, Peoples R China
ABSTRACT: The origination of new genes contributes to phenotypic evolution in humans. Two major challenges in the study of new genes are the inference of gene ages and annotation of their protein-coding potential. To tackle these challenges, we created GenTree, an integrated online database that compiles age inferences from three major methods together with functional genomic data for new genes. Genome-wide comparison of the age inference methods revealed that the synteny-based pipeline (SBP) is most suited for recently duplicated genes, whereas the protein-family-based methods are useful for ancient genes.
[more...]
Use: pFind



PTMiner: Localization and Quality Control of Protein Modifications Detected in an Open Search and Its Application to Comprehensive Post-translational …
MOLECULAR & CELLULAR PROTEOMICS. 2019. An, ZW et al. Chinese Acad Sci, Acad Math & Syst Sci, Natl Ctr Math & Interdisciplinary Sci, Key Lab Random Complex Struct & Data Sci, Beijing 100190, Peoples R China.
ABSTRACT: The open (mass tolerant) search of tandem mass spectra of peptides shows great potential in the comprehensive detection of post-translational modifications (PTMs) in shotgun proteomics. However, this search strategy has not been widely used by the community, and one bottleneck of it is the lack of appropriate algorithms for automated and reliable post-processing of the coarse and error-prone search results. Here we present PTMiner, a software tool for confident filtering and localization of modifications (mass shifts) detected in an open search.
[more...]
Use: pParse; pFind



O-GlcNAcylation of Thr12/Ser56 in short-form O-GlcNAc transferase (sOGT) regulates its substrate selectivity
Journal of Biological chemistry. 2019. Liu, L et al. Nankai Univ, Coll Pharm, Tianjin 300353, Peoples R China.
ABSTRACT: O-GlcNAcylation is a ubiquitous protein glycosylation playing different roles on variant proteins. O-GlcNAc transferase (OGT) is the unique enzyme responsible for the sugar addition to nucleocytoplasmic proteins. Recently, multiple O-GlcNAc sites have been observed on short-form OGT (sOGT) and nucleocytoplasmic OGT (ncOGT), both of which locate in the nucleus and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser(389) in ncOGT (1036 amino acids) affects its nuclear translocation in HeLa cells. To date, the major O-GlcNAcylation sites and their roles in sOGT remain unknown.
[more...]
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Role of human Keap1 S53 and S293 residues in modulating the binding of Keap1 to Nrf2
Biochimie. 2019. Wei, Shuangshuang et al. Hainan Univ, 817 Nongke Lou,58 Peoples Rd, Haikou 570228, Hainan, Peoples R China
ABSTRACT: Keap1 is deemed as a suppressor of Nrf2 in cytoplasm by sequestrating Nrf2 to proteolysis as an adapter of the Cul3-Rbx1 E3 ubiquitin ligase complex. In the study, it was proposed that post-translational modification might affect the interaction between Nrf2 and Keap1, and the profiles of the phosphorylation of amino acid residues of Keap1 and its effects on the binding of Keap1 to Nrf2 was investigated. A mass spectrometry analysis revealed that S53 and 5293 were phosphorylated upon an oxidative stress.
[more...]
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Isotopic labeling and quantitative proteomics of acetylation on histones and beyond
Mass Spectrometry of Proteins: Methods and Protocols. 2019. Lund, Peder J et al. Epigenetics Institute, Department of Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
ABSTRACT: Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification.
[more...]
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Skeletal muscle proteome analysis provides insights on high altitude adaptation of yaks
Molecular Biology Reports. 2019. Wen, WT et al. Sichuan Univ, Coll Life Sci, Minist Educ, Key Lab Bioresources & Ecoenvironm, Chengdu 610064, Sichuan, Peoples R China.
ABSTRACT: The differences in proteome profile of longissimus thoracis (LT) muscles of yak (Bos grunniens) and cattle (Bos taurus) were investigated employing isobaric tag for relative and absolute quantification (iTRAQ) approach to identify differentially expressed proteins and to understand the cellular level adaptations of yaks to high altitudes. Fifty-two proteins were differentially expressed in the two species, among which 20 were up-regulated and 32 were down-regulated in yaks. Gene ontology (GO) annotation revealed that most of the differentially expressed proteins were involved in the molecular function of protein binding, catalytic activity, and structural activity.
[more...]
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An integrated proteomic and glycoproteomic study for differences on glycosylation occupancy in rheumatoid arthritis
Analytical and Bioanalytical Chemistry. 2019. Li, X et al. Georgia State Univ, Ctr Diagnost & Therapeut, 50 Decatur St SE, Atlanta, GA 30303 USA.
ABSTRACT: Rheumatoid arthritis (RA) is an autoimmune disease in which certain immune cells are dysfunctional and attack their own healthy tissues. There has been great difficulty in finding an accurate and efficient method for the diagnosis of early-stage RA. The present shortage of diagnostic methods leads to the rough treatments of the patients in the late stages, such as joint removing. Nowadays, there is an increasing focus on glyco-biomarkers discovery for malicious disease via MS-based strategy. In this study, we present an integrated proteomics and glycoproteomics approach to uncover the pathological changes of some RA-related glyco-biomarkers and glyco-checkpoints involved in the RA onset.
[more...]
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Rapid covalent-probe discovery by electrophile-fragment screening
Journal of the American Chemical Society. 2019. Resnick, E et al. Weizmann Inst Sci, Nancy & Stephen Grand Israel Natl Ctr Personalize, Dept Organ Chem, IL-7610001 Rehovot, Israel.
ABSTRACT: Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins.
[more...]
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Direct proteomic mapping of cysteine persulfidation
Antioxidants & redox signaling. 2019. Fu, L et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing Proteome Res Ctr, Beijing 102206, Peoples R China.
ABSTRACT: Aims: Cysteine persulfidation (also called sulfhydration or sulfuration) has emerged as a potential redox mechanism to regulate protein functions and diverse biological processes in hydrogen sulfide (H2S) signaling. Due to its intrinsically unstable nature, working with this modification has proven to be challenging. Although methodological progress has expanded the inventory of persulfidated proteins, there is a continued need to develop methods that can directly and unequivocally identify persulfidated cysteine residues in complex proteomes.
[more...]
Use: pFind; pQuant



A chemical strategy for protease substrate profiling
Cell Chemical Biology. 2019. Griswold, AR et al. Mem Sloan Kettering Canc Ctr, Weill Cornell Grad Sch Med Sci, Pharmacol Program, New York, NY 10065 USA.
ABSTRACT: The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position.
[more...]
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Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex
EMBO JOURNAL. 2019. Zhang, G et al. Univ Copenhagen, Novo Nordisk Fdn Ctr Prot Res, Fac Hlth & Med Sci, Copenhagen, Denmark.
ABSTRACT: Kinetochore localized Mad1 is essential for generating a "wait anaphase" signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod-Zw10-Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins.
[more...]
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Precision De Novo Peptide Sequencing Using Mirror Proteases of Ac-LysargiNase and Trypsin for Large-scale Proteomics*[S]
Technological Innovation and Resources. 2019. Hao Yang; Yan-Chang Li; Ming-Zhi Zhao et al. State Key Laboratory of Proteomics; Beijing Proteome Research Center; National Center for Protein Sciences Beijing; Beijing Institute of Lifeomics, Beijing 102206, China;Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education (Wuhan University), Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China;College of Life Sciences, Hebei University, Baoding 071002, China(X.P.); Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS; University of Chinese Academy of Sciences; Institute of Computing Technology, CAS, Beijing 100190, China
ABSTRACT: De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing.
[more...]
Use: pFind; pNovo



Assessing the relationship between mass window width and retention time scheduling on protein coverage for data-independent acquisition
Journal of the American Society for Mass Spectrometry. 2019. Li, WX et al. Yale Univ, Yale Canc Biol Inst, West Haven, CT 06516 USA.
ABSTRACT: Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity.
[more...]
Use: pFind



Open-pFind enhances the identification of missing proteins from human testis tissue
Journal of proteome research. 2019. Sun, JS et al. Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, Guangzhou 510275, Guangdong, Peoples R China.
ABSTRACT: In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples.
[more...]
Use: pFind; pDeep



Open search unveils modification patterns in formalin-fixed, paraffin-embedded thermo HCD and SCIEX TripleTOF shotgun proteomes
International Journal of Mass Spectrometry. 2019. Tabb, DL et al. Stellenbosch Univ, Dept Biomed Sci, Div Mol Biol & Human Genet, Bioinformat Unit,South African TB Bioinformat Ini, Cape Town, South Africa.
ABSTRACT: The application of database search algorithms with very wide precursor mass tolerances for the "Open Search" paradigm has brought new efforts at post-translational modification discovery in shotgun proteomes. This approach has motivated the acceleration of database search tools by incorporating fragment indexing features. In this report, we compare open searches and sequence tag searches of high-resolution tandem mass spectra to seek a common "palette" of modifications when analyzing multiple formalin-fixed, paraffin-embedded (FFPE) tissues from Thermo Q-Exactive and SCIEX TripleTOF instruments.
[more...]
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Size-dependent sub-proteome analysis of urinary exosomes
Analytical and Bioanalytical Chemistry. 2019. Guan, S et al. Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China.
ABSTRACT: Exosomes are cell-derived functional microparticles which exist in most body fluids. They carry abundant signaling molecules to transfer information between cells and microenvironment. Research on exosomes' heterogeneity and constitute variations has been a heated topic in recent years. In this work, size-dependent sub-proteome analysis of urinary exosomes was investigated by size exclusion chromatography (SEC) firstly. The particle size of urinary exosomes is distributed in four main ranges naturally.
[more...]
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A thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome
Analytica Chimica Acta. 2019. Cai, Y et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome.
[more...]
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Zebrafish prmt5 arginine methyltransferase is essential for germ cell development
DEVELOPMENT. 2019. Zhu, JJ et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Hubei, Peoples R China.
ABSTRACT: Protein arginine methyltransferase 5 (Prmt5), a type II arginine methyltransferase, symmetrically dimethylates arginine in nuclear and cytoplasmic proteins. Prmt5 is involved in a variety of cellular processes, including ribosome biogenesis, cellular differentiation, germ cell development and tumorigenesis. However, the mechanisms by which prmt5 influences cellular processes have remained unclear. Here, prmt5 loss in zebrafish led to the expression of an infertile male phenotype due to a reduction in germ cell number, an increase in germ cell apoptosis and the failure of gonads to differentiate into normal testes or ovaries.
[more...]
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Mass spectrometry--driven exploration reveals nuances of neoepitope-driven tumor rejection
JCI Insight. 2019. Ebrahimi-Nik, H et al. Univ Connecticut, Sch Med, Dept Immunol, Farmington, CT 06032 USA.
ABSTRACT: Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time to our knowledge, we use a combination of genomics, unbiased discovery mass spectrometry (MS) immunopeptidomics, and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection-mediating neoepitopes (TRMNs).
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Quick and clean: Cracking sentences encoded in E. coli by LC--MS/MS, de novo sequencing, and dictionary search
EuPA Open Proteomics. 2019. Niu, Lili et al. Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
ABSTRACT: In this study, we faced the challenge of deciphering a protein that has been designed and expressed by E. coli in such a way that the amino acid sequence encodes two concatenated English sentences. The letters 'O' and 'U' in the sentence are both replaced by 'K' in the protein. The sequence cannot be found online and carried to-be-discovered modifications. With limited information in hand, to solve the challenge, we developed a workflow consisting of bottom-up proteomics, de novo sequencing and a bioinformatics pipeline for data processing and searching for frequently appearing words.
[more...]
Use: pNovo; pFind



Nitration-induced ubiquitination and degradation control quality of ERK1
Biochemical Journal. 2019. Zhang, YY et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol Dev Biol, 1 West Beichen Rd, Beijing 100101, Peoples R China.
ABSTRACT: The mitogen-activated protein kinase ERK1/2 (ERKs, extracellular-regulated protein kinases) plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP (C-terminal of Hsp70-interacting protein)-mediated ubiquitination and degradation.
[more...]
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Mutual regulation of receptor-like kinase SIT1 and B'$\kappa$-PP2A shapes the early response of rice to salt stress
The Plant Cell. 2019. Zhao, Ji-Long et al. 1; College of Life Science, Hebei Normal University, Hebei Key Laboratory of 7; Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology 8; of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, 9; Shijiazhuang, Hebei 050024, China
ABSTRACT: The receptor-like kinase SIT1 acts as a sensor in rice (Oryza sativa) roots, relaying salt stress signals via elevated kinase activity to enhance salt sensitivity. Here, we demonstrate that Protein Phosphatase 2A (PP2A) regulatory subunit B'kappa constrains SIT1 activity under salt stress. B'kappa-PP2A deactivates SIT1 directly by dephosphorylating the kinase at Thr515/516, a salt-induced phosphorylation site in the activation loop that is essential for SIT1 activity. B'kappa overexpression suppresses the salt sensitivity of rice plants expressing high levels of SIT1, thereby contributing to salt tolerance.
[more...]
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A multi-parallel N-glycopeptide enrichment strategy for high-throughput and in-depth mapping of the N-glycoproteome in metastatic human hepatocellular carcinoma cell lines
Talanta. 2019. Jiang, BY et al. Fudan Univ, Peoples Hosp Shanghai 5, Shanghai 200433, Peoples R China.
ABSTRACT: N-glycosylation is deeply involved in many biological processes, and approximately 50% of mammalian proteins are predicted to be glycosylated. Many large-scale studies have been carried out to reveal the glycosylation status involved in different physiological pathologies across species. However, the lack of a highly specific and high throughput N-glycosylated enrichment method not only results in extended time requirements but also limits the depth of mapping when handling a large number of samples.
[more...]
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Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4, 5-bisphosphate
Journal of Biological Chemistry. 2019. Roy, Neeladri Sekhar et al. Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT: Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2).
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Insights into the assembly and architecture of a Staufen-mediated mRNA decay (SMD)-competent mRNP
Nature Communications. 2019. Gowravaram, M et al. Free Univ Berlin, Inst Chem & Biochem, Takustr 6, D-14195 Berlin, Germany.
ABSTRACT: The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3'-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD.
[more...]
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Mechanism of protein cleavage at asparagine leading to protein–protein cross-links
Biochemical Journal. 2019. Friedrich, MG et al. Univ Wollongong, Illawarra Hlth & Med Res Inst, Wollongong, NSW 2522, Australia.
ABSTRACT: Long-lived proteins (LLPs) are present in numerous tissues within the human body. With age, they deteriorate, often leading to the formation of irreversible modifications such as peptide bond cleavage and covalent cross-linking. Currently understanding of the mechanism of formation of these cross-links is limited. As part of an ongoing study, proteomics was used to characterise sites of novel covalent cross-linking in the human lens. In this process, Lys residues were found cross-linked to C-terminal aspartates that had been present in the original protein as Asn residues.
[more...]
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Smart cutter: an efficient strategy for increasing the coverage of chemical cross-linking analysis
Analytical Chemistry. 2019. Zhao, LL et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT: Chemical cross-linking combined with mass spectrometry (CXMS) has emerged as a powerful tool to study protein structure, conformation, and protein-protein interactions (PPIs). Until now, most cross-linked peptides were generated by using commercial cross-linkers, such as DSS, BS3, and DSSO, which react with the primary amino groups of the lysine residues of proteins. However, trypsin, the most commonly used proteolytic enzyme, cannot cleave the C-terminus of a linked lysine, making the obtained cross-linked peptides longer than common peptides and unfavorable for MS identification and data searching.
[more...]
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LECT2, a ligand for Tie1, plays a crucial role in liver fibrogenesis
Cell. 2019. Xu, M et al. Southern Med Univ, Nanfang Hosp, Dept Pathol, Guangzhou 510515, Guangdong, Peoples R China.
ABSTRACT: Liver fibrosis is a very common condition seen in millions of patients with various liver diseases, and yet no effective treatments are available owing to poorly characterized molecular pathogenesis. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2) is a functional ligand of Tie1, a poorly characterized endothelial cell (EC)-specific orphan receptor. Upon binding to Tie1, LECT2 interrupts Tie1/Tie2 heterodimerization, facilitates Tie2/Tie2 homodimerization, activates PPAR signaling, and inhibits the migration and tube formations of EC.
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Global redox proteome and phosphoproteome analysis reveals redox switch in Akt
Nature Communications. 2019. Su, ZD et al. Univ Sydney, Charles Perkins Ctr, Sydney, NSW 2006, Australia.
ABSTRACT: Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications.
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A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation
Nature cell biology. 2019. Zhao, K et al. Chinese Acad Sci, Inst Biophys, CAS Ctr Excellence Biomacromol, Key Lab RNA Biol, Beijing, Peoples R China.
ABSTRACT: The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing.
[more...]
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Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16
Food Chemistry. 2019. Ge, JZ et al. Chinese Acad Agr Sci, Minist Agr, Feed Res Inst, Key Lab Feed Biotechnol, 12 Zhongguancun South St, Beijing 100081, Peoples R China.
ABSTRACT: Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 degrees C and pH 7.0, and was stable at 30 degrees C and pH 6.0-9.0 for 1 h, with a half-life of 15 min at 40 degrees C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 degrees C increased approximately 48-fold, and was stable at 30 degrees C and pH 3.0-12.0 for 1 h.
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Mass spectrometric determination of disulfide bonds and free cysteine in grass carp IgM isoforms
Fish & Shellfish Immunology. 2019. Su, YL et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, 152 Luoyu Rd, Wuhan 430079, Hubei, Peoples R China.
ABSTRACT: Disulfide bonds are fundamental in establishing Ig structure and maintaining Ig biological function. Here, we analysed disulfide bonds and free cysteine in three grass carp IgM isoforms (monomeric, dimeric/trimeric, and tetrameric IgM) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The results revealed that Cys(574) residue status at the C-terminal tail differed substantially in monomeric IgM in comparison with polymeric IgM, Cys(574) was found as free thiol in monomeric IgM, while it formed disulfide linkages in dimeric/trimeric and tetrameric IgM.
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CasX enzymes comprise a distinct family of RNA-guided genome editors
Nature. 2019. Liu, JJ et al. Univ Calif Berkeley, Dept Mol & Cell Biol, 229 Stanley Hall, Berkeley, CA 94720 USA.
ABSTRACT: The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification.
[more...]
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Structural basis of TFIIH activation for nucleotide excision repair
Nature Communications. 2019. Kokic, G et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double-and single-stranded DNA, consistent with their translocase and helicase activities, respectively.
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An integrated workflow for crosslinking mass spectrometry
Molecular systems biology. 2019. Mendes, ML et al. Tech Univ Berlin, Inst Biotechnol, Bioaralyt, Berlin, Germany.
ABSTRACT: We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
Use: pLink



RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
Nature Structural & Molecular Biology. 2019. Zhang, Q et al. Monash Univ, Biomed Discovery Inst, Dept Biochem & Mol Biol, Fac Med Nursing & Hlth Sci, Clayton, Vic, Australia.
ABSTRACT: Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identity during development in multicellular organisms by marking repressed genes and chromatin domains. In addition to four core subunits, PRC2 comprises multiple accessory subunits that vary in their composition during cellular differentiation and define two major holo-PRC2 complexes: PRC2.1 and PRC2.2. PRC2 binds to RNA, which inhibits its enzymatic activity, but the mechanism of RNA-mediated inhibition of holo-PRC2 is poorly understood.
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Improving mass spectrometry analysis of protein structures with arginine-selective chemical cross-linkers
Nature Communications. 2019. Jones, AX et al. Peking Univ, Beijing Natl Lab Mol Sci, Key Lab Bioorgan Chem & Mol Engn, Minist Educ,Dept Chem Biol,Coll Chem & Mol Engn,S, Beijing 100871, Peoples R China.
ABSTRACT: Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO.
[more...]
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Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly
Nature communications. 2019. Cuellar, J et al. Campus Univ Autonoma Madrid, Ctr Nacl Biotecnol, Madrid 28049, Spain.
ABSTRACT: The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, beta-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 angstrom) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings.
[more...]
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An integrated approach for determining a protein–protein binding interface in solution and an evaluation of hydrogen–deuterium exchange kinetics for adjudicating …
Analytical Chemistry. 2019. Zhang, MM et al. Washington Univ, Dept Chem, St Louis, MO 63130 USA.
ABSTRACT: We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its alpha-receptor, IL-7R alpha, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7R alpha but shows no differential evidence of binding-induced protection or remote conformational change.
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Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate
PROTEIN & CELL. 2019. Zhou, DJ et al. Peking Union Med Coll, Grad Sch, Beijing 100730, Peoples R China.
ABSTRACT: Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA.
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The molecular architecture of native BBSome obtained by an integrated structural approach
Structure. 2019. Chou, HT et al. Harvard Med Sch, Dept Cell Biol, Boston, MA 02115 USA.
ABSTRACT: The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete C alpha model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-angstrom resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data.
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The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants
Nature communications. 2019. Kesten, C et al. Univ Melbourne, Sch Biosci, Parkville, Vic 3010, Australia.
ABSTRACT: Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions.
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Near-atomic structure of a giant virus
Nature Communications. 2019. Fang, QL et al. Purdue Univ, Dept Biol Sci, W Lafayette, IN 47907 USA.
ABSTRACT: Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 angstrom resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types.
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Structural basis of poxvirus transcription: vaccinia RNA polymerase complexes
CELL. 2019. Grimm, C et al. Univ Wurzburg, Dept Biochem, D-97074 Wurzburg, Germany.
ABSTRACT: Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryoEM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 angstrom resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNA(Gln).
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Structural basis of specific H2A K13/K15 ubiquitination by RNF168
Nature Communications. 2019. Horn, V et al. Leiden Univ, Leiden Inst Chem, Dept Macromol Biochem, POB 95022300 RA, Leiden, Netherlands.
ABSTRACT: Ubiquitination of chromatin by modification of histone H2A is a critical step in both regulation of DNA repair and regulation of cell fate. These very different outcomes depend on the selective modification of distinct lysine residues in H2A, each by a specific E3 ligase. While polycomb PRC1 complexes modify K119, resulting in gene silencing, the E3 ligase RNF168 modifies K13/15, which is a key event in the response to DNA double-strand breaks. The molecular origin of ubiquitination site specificity by these related E3 enzymes is one of the open questions in the field.
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Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome
Nature communications. 2019. Fau, SV et al. Inst Canc Res, Div Struct Biol, London SW3 6JB, England.
ABSTRACT: Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery.
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Mechanistic insights into the SNARE complex disassembly
SCIENCE ADVANCES. 2019. Huang, X et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, State Key Lab Membrane Biol, Beijing 100084, Peoples R China.
ABSTRACT: NSF (N-ethylmaleimide-sensitive factor) and alpha-SNAP (alpha-soluble NSF attachment protein) bind to the SNARE (soluble NSF attachment protein receptor) complex, the minimum machinery to mediate membrane fusion, to form a 20S complex, which disassembles the SNARE complex for reuse. We report the cryo-EM structures of the alpha-SNAP-SNARE subcomplex and the NSF-D1D2 domain in the 20S complex at 3.9- and 3.7-angstrom resolutions, respectively. Combined with the biochemical and electrophysiological analyses, we find that alpha-SNAPs use R116 through electrostatic interactions and L197 through hydrophobic interactions to apply force mainly on two positions of the VAMP protein to execute disassembly process.
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Phycobilisomes Harbor FNRL in Cyanobacteria
MBio. 2019. Liu, HJ et al. Washington Univ, Dept Biol, Campus Box 1137, St Louis, MO 63130 USA.
ABSTRACT: Cyanobacterial phycobilisomes (PBSs) are photosynthetic antenna complexes that harvest light energy and supply it to two reaction centers (RCs) where photochemistry starts. PBSs can be classified into two types, depending on the presence of allophycocyanin (APC): CpcG-PBS and Cod-PBS. Because the accurate protein composition of CpcL-PBS remains unclear, we describe here its isolation and characterization from the cyanobacterium Synechocystis sp. strain 6803. We found that ferredoxin-NADP oxidoreductase (or FNRL), an enzyme involved in both cyclic electron transport and the terminal step of the electron transport chain in oxygenic photosynthesis, is tightly associated with CpcL-PBS as well as with CpcG-PBS.
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The internal interaction in RBBP5 regulates assembly and activity of MLL1 methyltransferase complex
Nucleic acids research. 2019. Han, JM et al. Chinese Acad Sci, State Key Lab Mol Biol, Natl Ctr Prot Sci Shanghai, CAS Ctr Excellence Mol Cell Sci,Shanghai Inst Bio, 333 Haike Rd, Shanghai 201210, Peoples R China.
ABSTRACT: The Mixed Lineage Leukemia protein 1 (MLL1) plays an essential role in the maintenance of the histone H3 lysine 4 (H3K4) methylation status for gene expression during differentiation and development. The methyltransferase activity of MLL1 is regulated by three conserved core subunits, WDR5, RBBP5 and ASH2L. Here, we determined the structure of human RBBP5 and demonstrated its role in the assembly and regulation of the MLL1 complex. We identified an internal interaction between the WD40 propeller and the C-terminal distal region in RBBP5, which assisted the maintenance of the compact conformation of the MLL1 complex.
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Structural and functional analyses of the human PDH complex suggest a “division-of-labor” mechanism by local E1 and E3 clusters
STRUCTURE. 2019. Prajapati, S et al. Georg August Univ Gottingen, Gottingen Ctr Mol Biosci, Dept Mol Enzymol, Julia Lermontowa Weg 3, D-37077 Gottingen, Germany.
ABSTRACT: The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-A resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes.
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Heat shock protein 104 (HSP104) chaperones soluble Tau via a mechanism distinct from its disaggregase activity
Journal of Biological Chemistry. 2019. Zhang, X et al. Chinese Acad Sci, Shanghai Inst Organ Chem, Interdisciplinary Res Ctr Biol & Chem, 26 Qiuyue Rd, Shanghai 201210, Peoples R China.
ABSTRACT: Heat shock protein 104 (HSP104) is a conserved AAA+ protein disaggregase, can disassemble the toxic aggregates formed by different amyloid proteins, and is protective in various animal models associated with amyloid-related diseases. Extensive studies have attempted to elucidate how HSP104 disassembles the aggregated form of clients. Here, we found that HSP104 exhibits a potent holdase activity that does not require energy, prevents the soluble form of amyloid clients from aggregating, and differs from HSP104's disaggregase activity.
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The Vaccinia virion: Filling the gap between atomic and ultrastructure
PLOS Pathogens. 2019. Mirzakhanyan, Y et al. UC Irvine, Dept Mol Biol & Biochem, Irvine, CA 92697 USA.
ABSTRACT: We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins.
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Comprehensive detection of isopeptides between human tissue transglutaminase and gluten peptides
Nutrients. 2019. Lexhaller, B et al. Tech Univ Munich, Leibniz Inst Food Syst Biol, Lise Meitner Str 34, D-85354 Freising Weihenstephan, Germany.
ABSTRACT: Celiac disease (CD) is a chronic inflammation of the small intestine triggered by the ingestion of gluten in genetically predisposed individuals. Tissue transglutaminase (TG2) is a key factor in CD pathogenesis, because it catalyzes both the deamidation of specific glutamine residues and the formation of covalent N epsilon-(gamma-glutamyl)-lysine isopeptide crosslinks resulting in TG2-gluten peptide complexes. These complexes are thought to activate B cells causing the secretion of anti-TG2 autoantibodies that serve as diagnostic markers for CD, although their pathogenic role remains unclear.
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Evaluation of different stationary phases in the separation of inter-cross-linked peptides
Journal of proteome research. 2019. Fang, ZX et al. Univ Texas Arlington, Dept Chem & Biochem, Arlington, TX 76019 USA.
ABSTRACT: Chemical cross-linking coupled with mass spectrometry (MS) is becoming a routinely and widely used technique for depicting and constructing protein structures and protein interaction networks. One major challenge for cross-linking/MS is the determination of informative low abundant inter-cross-linked products, generated within a sample of high complexity. A C18 stationary phase is the conventional means for reversed-phase (RP) separation of inter-cross-linked peptides. Various RP stationary phases, which provide different selectivities and retentions, have been developed as alternatives to C18 stationary phases.
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Large conformation shifts of Vibrio cholerae VqmA dimer in the absence of target DNA provide insight into DNA-binding mechanisms of LuxR-type receptors
Biochemical and biophysical research communications. 2019. Wu, H et al. Chinese Acad Sci, Shanghai Inst Appl Phys, Shanghai 201800, Peoples R China.
ABSTRACT: Quorum sensing regulates the biofilm formation and expression of virulence factors in Vibrio cholerae, an obligate human pathogen that continues to imperil human health. Cytoplasmic transcription factor VqmA is a LuxR-type receptor ubiquitous in the Vibrio genus and one vibriophage VP882 and plays an important role in V. cholerae pathogenicity. Here we presented the X-ray crystal structure of V. cholerae VqmA-DPO complex and compared it with the previously determined VqmA-DPO-DNA complex. To our knowledge, this is the first report on the crystal structures of the same LuxR-type receptor with two conformations of binding to DNA and not binding to DNA.
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Xolik: finding cross-linked peptides with maximum paired scores in linear time
Bioinformatics. 2019. Dai, JA et al. Hong Kong Univ Sci & Technol, Dept Elect & Comp Engn, Hong Kong, Peoples R China.
ABSTRACT: Motivation: Cross-linking technique coupled with mass spectrometry (MS) is widely used in the analysis of protein structures and protein-protein interactions. In order to identify cross-linked peptides from MS data, we need to consider all pairwise combinations of peptides, which is computationally prohibitive when the sequence database is large. To alleviate this problem, some heuristic screening strategies are used to reduce the number of peptide pairs during the identification. However, heuristic screening strategies may miss some true cross-linked peptides.
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Characterization of PPIB interaction in the P3H1 ternary complex and implications for its pathological mutations
Cellular and Molecular Life Sciences. 2019. Wu, JW et al. Shanghai Jiao Tong Univ, Sch Med, Dept Pathophysiol, Shanghai Tongren Hosp,Fac Basic Med,Hongqiao Int, Shanghai 200025, Peoples R China.
ABSTRACT: The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex.
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Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4, 5 …
Journal of Biological Chemistry. 2019. Roy, NS et al. NCI, Lab Cellular & Mol Biol, Ctr Canc Res, NIH, Frederick, MD 21702 USA.
ABSTRACT: Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2).
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Molecular architecture of the Bardet–Biedl syndrome protein 2-7-9 subcomplex
Journal of Biological Chemistry. 2019. Ludlam, WG et al. Brigham Young Univ, Dept Chem & Biochem, Provo, UT 84602 USA.
ABSTRACT: Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9.
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Crystal structure and activation mechanism of DR3 death domain
The Federation of European Biochemical Societies Journal. 2019. Yin, XY et al. Univ Sci & Technol China, Med Ctr, Hefei, Anhui, Peoples R China.
ABSTRACT: Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 angstrom resolutions, respectively. The mouse DR3 DD adopts a classical six-helix bundle structure while human DR3 DD displays an extended fold. Though there is one-amino-acid difference in the linker between maltose-binding protein (MBP) tag and DR3 DD, according to our self-interaction analysis, the hydrophobic interface discovered in MBP-hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction.
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Discovery of Interacting Proteins of ABA Receptor PYL5 via Covalent Chemical Capture
ACS Chemical Biology. 2019. Zheng, QZ et al. Tsinghua Univ, Dept Chem, Ctr Basic Mol Sci, Beijing 100084, Peoples R China.
ABSTRACT: Abscisic acid (ABA) is a key phytohormone with diverse functions in plants, and its signal transduction is mainly mediated by ABA receptors termed PYR/PYL/RCARs (hereafter referred to as PYLs) through the PYLs-PP2Cs-SnRK2s regulatory systems. However, the model failed to account for the roles of some important known regulators of ABA physiology. Given the central role of PYLs in ABA signal transduction, we therefore speculated that ABA receptors PYLs might be involved in regulatory pathways other than PP2Cs.
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Identification of Cross-linked Peptides Using Isotopomeric Cross-linkers
Journal of The American Society for Mass Spectrometry. 2019. Luo, J et al. Inst Syst Biol, 401 Terry Ave North, Seattle, WA 98109 USA.
ABSTRACT: Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information.
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Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4, 5-bisphosphate
Journal of Biological Chemistry. 2019. Roy, NS et al. NCI, Lab Cellular & Mol Biol, Ctr Canc Res, NIH, Frederick, MD 21702 USA.
ABSTRACT: Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2).
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An integrated approach for determining a protein--protein binding interface in solution and an evaluation of hydrogen--deuterium exchange kinetics for adjudicating candidate docking models
Analytical Chemistry. 2019. Zhang, MM et al. Washington Univ, Dept Chem, St Louis, MO 63130 USA.
ABSTRACT: We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its alpha-receptor, IL-7R alpha, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7R alpha but shows no differential evidence of binding-induced protection or remote conformational change.
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Smart Cutter: An Efficient Strategy for Increasing the Coverage of Chemical Cross-Linking Analysis
Analytical Chemistry. 2019. Zhao, LL et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT: Chemical cross-linking combined with mass spectrometry (CXMS) has emerged as a powerful tool to study protein structure, conformation, and protein-protein interactions (PPIs). Until now, most cross-linked peptides were generated by using commercial cross-linkers, such as DSS, BS3, and DSSO, which react with the primary amino groups of the lysine residues of proteins. However, trypsin, the most commonly used proteolytic enzyme, cannot cleave the C-terminus of a linked lysine, making the obtained cross-linked peptides longer than common peptides and unfavorable for MS identification and data searching.
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Mass spectrometry--based molecular mapping of native FXIIIa cross-links in insoluble fibrin clots
Journal of Biological Chemistry. 2019. Schmitt, LR et al. Univ Colorado, Sch Med, Dept Biochem & Mol Genet, Aurora, CO 80045 USA.
ABSTRACT: The roles of factor XIIIa-specific cross-links in thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these cross-links could inform the development of therapeutic strategies to prevent the sequelae of thromboembolism. Here, we present an MS-based method to map native factor XIIIa cross-links in the insoluble matrix component of whole-blood or plasma-fibrin clots and in in vivo thrombi. Using a chaotrope-insoluble digestion method and quantitative cross-linking MS, we identified the previously mapped fibrinogen peptides that are responsible for covalent D-dimer association, as well as dozens of novel cross-links in the C region of fibrinogen .
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Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination
Molecular Cell. 2019. Valencia-Sanchez, MI et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, Skirball Inst Biomol Med, New York, NY 10016 USA.
ABSTRACT: The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Doti L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 angstrom and 4.9 angstrom, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub.
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单程直驱超高压纳升泵的研制与评价
色谱. 2019. 杨三东 et al. 大连依利特分析仪器有限公司, 辽宁 大连 116023,大连市色谱工程技术研究中心, 辽宁 大连 116023
ABSTRACT: With the development of life science, nano liquid chromatography systems are being involved in various applications in the field of biochemical analysis. Being one of the key components in the system, the nano flow rate pump directly affected the accuracy and repeatability of the analysis. Based on two high precision direct-driven motors and a ten-port switch valve, a single stroke direct-driven ultrahigh pressure nano pump was developed. The results show that the accuracy of the flow rate and stability were better than 1% and 0.7%, respectively, at 500 nL/min.
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Antioxidative effects and mechanism study of bioactive peptides from defatted walnut (Juglans regia L.) meal hydrolysate
Journal of Agricultural and Food Chemistry. 2019. Sheng, JY et al. Huazhong Univ Sci & Technol, Coll Life Sci & Technol, Natl Engn Res Ctr Nanomed, Wuhan 430074, Hubei, Peoples R China.
ABSTRACT: The peptide components of defatted walnut (Juglans regia L.) meal hydrolysate (DWMH) remain unclear, hindering the investigation of biological mechanisms and exploitation of bioactive peptides. The present study aims to identify the peptide composition of DWMH, followed by to evaluate in vitro antioxidant effects of selected peptides and investigate mechanisms of antioxidative effect. First, more than 1 000 peptides were identified by de novo sequencing in DWMH. Subsequently, a scoring method was established to select promising bioactive peptides by structure based screening.
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Use: pNovo; pXtract; pBuild; pParse



TagGraph reveals vast protein modification landscapes from large tandem mass spectrometry datasets
Nature Biotechnology. 2019. Devabhaktuni, A et al. Stanford Univ, Stanford Sch Med, Dept Chem & Syst Biol, Stanford, CA 94305 USA.
ABSTRACT: Although mass spectrometry is well suited to identifying thousands of potential protein post-translational modifications (PTMs), it has historically been biased towards just a few. To measure the entire set of PTMs across diverse proteomes, software must overcome the dual challenges of covering enormous search spaces and distinguishing correct from incorrect spectrum interpretations. Here, we describe TagGraph, a computational tool that overcomes both challenges with an unrestricted string-based search method that is as much as 350-fold faster than existing approaches, and a probabilistic validation model that we optimized for PTM assignments.
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Let me infuse this for you--A way to solve the first YPIC challenge
EuPA Open Proteomics. 2019. Eggers, Britta et al. Ruhr University Bochum, Faculty of Medicine, Medizinisches Proteom-Center, Gesundheitscampus 4, D-44801, Bochum, Germany
ABSTRACT: In a common proteomics analysis today, the origins of our sample in the vial are known and therefore a database dependent approach to identify the containing peptides can be used. The first YPIC challenge though provided us with 19 synthetic peptides, which together formed an English sentence. For the identification of these peptides, a de-novo approach was used, which brought us together with an internet search engine to the hidden sentence. But only having the sentence was not sufficient for us, we also wanted to identify as many as possible of the spectra in our data.
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SpotLight Proteomics—A IgG-Enrichment Phenotype Profiling Approach with Clinical Implications
International Journal of Molecular Sciences. 2019. Lundstrom, SL et al. Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 1, S-17177 Stockholm, Sweden.
ABSTRACT: Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (Lofgrens and non-Lofgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated.
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An integrated strategy for high-sensitive and multi-level glycoproteome analysis from low micrograms of protein samples
JOURNAL OF CHROMATOGRAPHY A. 2019. Gao, WN et al. Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China.
ABSTRACT: Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material.
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Use: pParse; pGlyco



Comparative glycoproteomic profiling of human body fluid between healthy controls and patients with papillary thyroid carcinoma
Journal of proteome research. 2019. Zhang, Y et al. Sichuan Univ, Key Lab Transplant Engn & Immunol, West China Washington Mitochondria & Metab Res Ct, West China Hosp,MOH, Chengdu 610041, Peoples R China.
ABSTRACT: Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer among women worldwide. It is confirmed mainly by fine-needle aspiration biopsy (FNAB), an invasive diagnostic method. The key proteins responsible for thyroid hormone biosynthesis are glycosylated. Hence, changes in site-specific glycosylation are associated with thyroid cancer. Integrated quantitative proteomic and glycoproteomic analyses of body fluids from patients with PTC may identify potential noninvasive biomarkers, improve diagnostic accuracy, and elucidate the basic mechanisms of tumor development.
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Use: pGlyco



Glyco-CPLL: an integrated method for in-depth and comprehensive N-glycoproteome profiling of human plasma
Journal of proteome research. 2019. Zhang, Y et al. Sichuan Univ, Chengdu, Peoples R China.
ABSTRACT: N-glycoproteins are involved in various biological processes. Certain distinctive glycoforms on specific glycoproteins enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential for a new biomarker discovery. The absence of suitable analytical methods for in-depth and large-scale analyses of low-abundance plasma glycoproteins makes it challenging to investigate the role of glycosylation. In this study, we developed an integrated method termed Glyco-CPLL, which integrates combinatorial peptide ligand libraries, high-pH reversed-phase prefractionation, hydrophilic interaction chromatography, trypsin and PNGase F digestion, shotgun proteomics, and various analysis software (MaxQuant and pGlyco2.0) for the low-abundance plasma glycoproteomic profiling.
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N-glycopeptide signatures of IgA2 in serum from patients with hepatitis B virus-related liver diseases
Molecular & Cellular Proteomics. 2019. Zhang, Shu et al. Fudan Univ, Inst Biomed Sci, Shanghai 200032, Peoples R China; Fudan Univ, Key Lab Carcinogenesis & Canc Invas, Minist Educ, Shanghai 200032, Peoples R China; Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai 200032, Peoples R China; Fudan Univ, Zhongshan Hosp, Liver Canc Inst, Shanghai 200032, Peoples R China
ABSTRACT: N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40-55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on O-18/O-16 C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC).
[more...]
Use: pGlyco; pQuant



Proteomics analysis of site-specific glycoforms by a virtual multistage mass spectrometry method
Analytica Chimica Acta. 2019. Qin, HQ et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Determination of site-specific glycoforms is the key to reveal the micro-heterogeneity of protein glycosylation at proteome level. Herein, we presented an integrated virtual multistage MS strategy to identify intact glycopeptides, which allowed the determination of site-specific glycoforms. In this strategy, the enzymatically de-glycosylated peptides and intact glycopeptides were mixed and analyzed in the same LC-MS/MS run. The acquired MS2 spectra of intact glycopeptides allowed determination of the glycans, and the MS2 spectra of the de-glycosylated peptides enabled the identification of peptide backbone sequences.
[more...]
Use: pGlyco



Highly efficient analysis of glycoprotein sialylation in human serum by simultaneous quantification of glycosites and site-specific glycoforms
Journal of proteome research. 2019. Qin, HQ et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: Aberrant sialylation of glycoproteins is closely related to many malignant diseases, and analysis of sialylation has great potential to reveal the status of these diseases. However, in-depth analysis of sialylation is still challenging because of the high microheterogeneity of protein glycosylation, as well as the low abundance of sialylated glycopeptides (SGPs). Herein, an integrated strategy was fabricated for the detailed characterization of glycoprotein sialylation on the levels of glycosites and site-specific glycoforms by employing the SGP enrichment method.
[more...]
Use: pGlyco; pQuant



A practical approach to enrich intact tryptic N-glycopeptides through size exclusion chromatography and hydrophilicity (SELIC) using an acrylamide-agarose composite gel system
Analytica Chimica Acta. 2019. Zhao, T et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Increasing researches proved that abnormal glycosylation is strongly correlated with many diseases. Specially, site-specific glycosylation and its associated heterogeneity are closely related to the function and activity of the glycoprotein. However, intact N-glycopeptide analysis still faces great challenges because the presence of highly abundant non-glycosylated peptides would suppress the ionization of lowly abundant glycopeptides. In the present study, we developed a practical intact tryptic N-glycopeptide enrichment method using acrylamide-agarose composite gel that combined the size exclusion chromatography and hydrophilic (named SELIC) effects, aimed to remove the detergent rapidly and effectively, as well as enrich intact N-glycopeptides while extracting peptides.
[more...]
Use: pGlyco



N-glycopeptide Signatures of IgA2 in Serum from Patients with Hepatitis B Virus-related Liver Diseases*[S]
Molecular & Cellular Proteomics. 2019. Zhang, S et al. Fudan Univ, Zhongshan Hosp, Liver Canc Inst, Shanghai 200032, Peoples R China.
ABSTRACT: N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40-55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on O-18/O-16 C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC).
[more...]
Use: pGlyco; pQuant



SPECTRUM--A MATLAB toolbox for proteoform identification from top-down proteomics data
Scientific reports. 2019. Basharat, Abdul Rehman et al. Lahore Univ Management Sci, Dept Biol, Biomed Informat Res Lab, Lahore, Pakistan
ABSTRACT: Top-Down Proteomics (TDP) is an emerging proteomics protocol that involves identification, characterization, and quantitation of intact proteins using high-resolution mass spectrometry. TDP has an edge over other proteomics protocols in that it allows for: (i) accurate measurement of intact protein mass, (ii) high sequence coverage, and (iii) enhanced identification of post-translational modifications (PTMs). However, the complexity of TDP spectra poses a significant impediment to protein search and PTM characterization.
[more...]
Use: pTop



Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning
Nature methods. 2019. Gessulat, S et al. Tech Univ Munich, Chair Prote & Bioanalyt, Freising Weihenstephan, Germany.
ABSTRACT: In mass-spectrometry-based proteomics, the identification and quantification of peptides and proteins heavily rely on sequence database searching or spectral library matching. The lack of accurate predictive models for fragment ion intensities impairs the realization of the full potential of these approaches. Here, we extended the ProteomeTools synthetic peptide library to 550,000 tryptic peptides and 21 million high-quality tandem mass spectra. We trained a deep neural network, termed Prosit, resulting in chromatographic retention time and fragment ion intensity predictions that exceed the quality of the experimental data.
[more...]
Use: pDeep



Prediction of lc-ms/ms properties of peptides from sequence by deep learning*[s]
Molecular & Cellular Proteomics. 2019. Guan, SH et al. Univ Waterloo, David R Cheriton Sch Comp Sci, Waterloo, ON N2L 3G1, Canada.
ABSTRACT: Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications.
[more...]
Use: pDeep



MS2CNN: predicting MS/MS spectrum based on protein sequence using deep convolutional neural networks
BMC genomics. 2019. Lin, YM et al. Natl Chengchi Univ, Dept Comp Sci, Taipei 11605, Taiwan.
ABSTRACT: Background: Tandem mass spectrometry allows biologists to identify and quantify protein samples in the form of digested peptide sequences. When performing peptide identification, spectral library search is more sensitive than traditional database search but is limited to peptides that have been previously identified. An accurate tandem mass spectrum prediction tool is thus crucial in expanding the peptide space and increasing the coverage of spectral library search. Results: We propose (MSCNN)-C-2, a non-linear regression model based on deep convolutional neural networks, a deep learning algorithm.
[more...]
Use: pDeep



Prediction of LC-MS/MS Properties of Peptides from Sequence by Deep Learning*[S]
Molecular & Cellular Proteomics. 2019. Guan, SH et al. Univ Waterloo, David R Cheriton Sch Comp Sci, Waterloo, ON N2L 3G1, Canada.
ABSTRACT: Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications.
[more...]
Use: pDeep




2018




S-nitrosylation targets GSNO reductase for selective autophagy during hypoxia responses in plants
Molecular cell. 2018. Zhan, Ni et al. Chinese Acad Sci, Inst Genet & Dev Biol, CAS Ctr Excellence Mol Plant Sci, Beijing 100101, Peoples R China; Chinese Acad Sci, State Key Lab Plant Genom, Beijing 100101, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China
ABSTRACT: Nitric oxide (NO) regulates diverse cellular signaling through S-nitrosylation of specific Cys residues of target proteins. The intracellular level of S-nitrosoglutathione (GSNO), a major bioactive NO species, is regulated by GSNO reductase (GSNOR), a highly conserved master regulator of NO signaling. However, little is known about how the activity of GSNOR is regulated. Here, we show that S-nitrosylation induces selective autophagy of Arabidopsis GSNOR1 during hypoxia responses. S-nitrosylation of GSNOR1 at Cys-10 induces conformational changes, exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) accessible by autophagy machinery.
[more...]
Use: pFind



Proteomic analysis and NIR-II imaging of MCM2 protein in hepatocellular carcinoma
Journal of proteome research. 2018. Yang, Jing et al. Wuhan Univ, Sch Pharmaceut Sci, Zhongnan Hosp, Wuhan 430071, Peoples R China; Tibet Univ, Coll Med, Lasa 850000, Peoples R China; Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China; Wuhan Univ, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery M, State Key Lab Virol, Wuhan 430071, Peoples R China; Wuhan Univ, Hubei Prov Key Lab Dev Originated Dis, Wuhan 430071, Peoples R China; Wuhan Univ, Sch Basic Med Sci, Hubei Prov Engn & Technol Res Ctr Fluorinated Pha, Ctr Expt Basic Med Educ, Wuhan 430071, Peoples R China; Anhui Med Univ, Hefei 230032, Anhui, Peoples R China
ABSTRACT: Targeted therapy of hepatocellular carcinoma (HCC) is essential for improved therapies. Therefore, identification of key targets specifically to HCC is an urgent requirement. Herein, an iTRAQ-quantitative proteomic approach was employed to identify differentially expressed proteins in HCC tumor tissues. Of the upregulated tumor-related proteins, minichromosome maintenance 2 (MCM2), a DNA replication licensing factor, was one of the most significantly altered proteins, and its over expression was confirmed using tissue microarray.
[more...]
Use: pFind



Chemical proteomics reveals new targets of cysteine sulfinic acid reductase
Nature Chemical Biology. 2018. Akter, S et al. Scripps Res Inst, Dept Chem, Jupiter, FL 33458 USA.
ABSTRACT: Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation.
[more...]
Use: pFind; pQuant




Site-specific N-glycosylation on the AAV8 capsid protein
VIRUSES-BASEL. 2018. Aloor, A et al. Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30302 USA.
ABSTRACT: Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation.
[more...]
Use: pFind



Hydrogen-deuterium exchange coupled to top-and middle-down mass spectrometry reveals histone tail dynamics before and after nucleosome assembly
Structure. 2018. Karch, KR et al. Univ Penn, Dept Biochem & Mol Biophys, Perelman Sch Med, Philadelphia, PA 19104 USA.
ABSTRACT: Until recently, a major limitation of hydrogen-deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization was limited to the length of the peptide generated during proteolysis. However, electron transfer dissociation (ETD) has been shown to preserve deuterium label in the gas phase, enabling better resolution. To date, this technology remains mostly limited to small, already well-characterized proteins. Here, we optimize, expand, and adapt HDX-MS tandem MS (MS/MS) capabilities to accommodate histone and nucleosomal complexes on top-down HDX-MS/MS and middle-down HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility.
[more...]
Use: pFind



Enrichment-Based Proteogenomics Identifies Microproteins, Missing Proteins, and Novel smORFs in Saccharomyces cerevisiae
Journal of Proteome Research. 2018. He, CT et al. Anhui Med Univ, Hefei 230032, Anhui, Peoples R China.
ABSTRACT: Microproteins are peptides composed of 100 amino acids (AA) or fewer, encoded by small open reading frames (smORFs). It has been demonstrated that microproteins participate in and regulate a wide range of functions in cells. However, the annotation and identification of microproteins is challenging in part owing to their low molecular weight, low abundancy, and hydrophobicity. These factors have led to the unannotation of smORFs in genome processing and have made their identification at the protein level difficult.
[more...]
Use: pFind



Genome annotation of a model diatom Phaeodactylum tricornutum using an integrated proteogenomic pipeline
Molecular Plant. 2018. Yang, MK et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Hubei, Peoples R China.
ABSTRACT: Diatoms comprise a diverse and ecologically important group of eukaryotic phytoplankton that significantly contributes to marine primary production and global carbon cycling. Phaeodactylum tricornutum is commonly used as a model organism for studying diatom biology. Although its genome was sequenced in 2008, a high-quality genome annotation is still not available for this diatom. Here we report the development of an integrated proteogenomic pipeline and its application for improved annotation of P.
[more...]
Use: pFind



Multiproteases combined with high-pH reverse-phase separation strategy verified fourteen missing proteins in human testis tissue
Journal of proteome research. 2018. Sun, Jinshuai et al. Demo Lab Thermofisher Sci China, Shanghai 200120, Peoples R China; Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Diver, Baoding 071002, Hebei, Peoples R China; Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China; Wuhan Univ, Sch Pharmaceut Sci, Key Lab Combinat Biosynth & Drug Discovery, Minist Educ, Wuhan 430072, Hubei, Peoples R China; Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, Guangdong Key Lab Plant Resources, Guangzhou 510275, Guangdong, Peoples R China
ABSTRACT: Subsequent to conducting the Chromosome-Centric Human Proteome Project, we have focused on human testis-enriched missing proteins (MPs) since 2015. For protein coverage to be enhanced, a multiprotease strategy was used for separation of samples by 10% SDS-PAGE. For the separating efficiency to be improved, a high-pH reverse phase (RP) separation strategy was applied to fractionate complex samples in this study. A total of 11,558 proteins was identified, which is the largest proteome data set for single human tissue sample so far.
[more...]
Use: pFind



N-linked glycopeptide identification based on open mass spectral library search
Biomed Research International. 2018. An, ZW et al. Chinese Acad Sci, Acad Math & Syst Sci, Natl Ctr Math & Interdisciplinary Sci, Key Lab Random Complex Struct & Data Sci, Beijing 100101, Peoples R China.
ABSTRACT: Confident characterization of intact glycopeptides is a challenging task in mass spectrometry-based glycoproteomics due to microheterogeneity of glycosylation, complexity of glycans, and insufficient fragmentation of peptide bones. Open mass spectral library search is a promising computational approach to peptide identification, but its potential in the identification of glycopeptides has not been fully explored. Here we present pMatchGlyco, a new spectral library search tool for intact N-linked glycopeptide identification using high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) data.
[more...]
Use: pFind; pParse; pGlyco



Digging for missing proteins using low-molecular-weight protein enrichment and a “mirror protease” strategy
Journal of Proteome Research. 2018. He, CT et al. Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, Guangdong Key Lab Plant Resources, Guangzhou 510275, Guangdong, Peoples R China.
ABSTRACT: In 2012, the Chromosome-centric Human Proteome Project (C-HPP) launched an investigation for missing proteins (MPs) to complete the Human Proteome Project (HPP). The majority of the MPs were distributed in low-molecular-weight (LMW) ranges, especially from 0 to 40 kDa. LMW protein identification is challenging, owing to their short length, low abundance, and hydrophobicity. Furthermore, many sequences from trypsin digestion are unlikely to yield detectable peptides or a reasonable quality of MS2 spectrum.
[more...]
Use: pFind



Proteomics investigation of the changes in serum proteins after high-and low-flux hemodialysis
RENAL FAILURE. 2018. Han, S et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT: Purpose: This study aimed to use proteomics methods to investigate the changes in serum protein levels after high- and low-flux hemodialysis (HD). Methods: Before and after HD, serum samples were obtained from two selected patients who were treated with a Polyflux 140H high-flux dialyzer and a Polyflux 14L low-flux dialyzer during two continuous therapy sessions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to identify the proteins. Results: A total of 212 and 203 serum proteins were identified after high-flux and low-flux HD, respectively.
[more...]
Use: pFind



Optimal settings of mass spectrometry open search strategy for higher confidence
Journal of Proteome Research. 2018. Li, DH et al. Jinan Univ, Inst Life & Hlth Engn, Coll Life Sci & Technol, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Guangdong, Peoples R China.
ABSTRACT: In most proteome mass spectrometry experiments, more than half of the mass spectra cannot be identified, mainly because of various modifications. The open search strategy allows for a larger precursor tolerance to utilize more spectra, especially those with post-translational modifications; however, thorough quality control based on independent information is lacking. Here, we used the "Suspicious Discovery Rate (SDR)" based on translatome sequencing (RNC-seq) as an independent source to reference the proteome open search results in steady-state cells.
[more...]
Use: pFind



Facile Cu (ii)-mediated conjugation of thioesters and thioacids to peptides and proteins under mild conditions
Organic & Biomolecular Chemistry. 2018. Sun, Y et al. Wuhan Univ, Sch Pharmaceut Sci, Zhongnan Hosp, State Key Lab Virol, Wuhan 430071, Hubei, Peoples R China.
ABSTRACT: The bioconjugation of peptide derivatives such as polypeptides, peptide-based probes and proteins is a vibrant area in many scientific fields. However, reports on metal-mediated chemical methods towards native peptides especially non-engineering protein modification under mild conditions are still limited. Herein, we describe a novel Cu(ii)-mediated strategy for the conjugation of thioesters/thioacids to peptides under mild conditions with high functional group tolerance. Based on this strategy, polypeptides, even peptide-based fluorescent probes, can be efficiently constructed.
[more...]
Use: pFind



Deep learning-based MSMS spectra reduction in support of running multiple protein search engines on cloud
2017 IEEE International Conference on Bioinformatics and Biomedicine (BIBM). 2018. Maabreh, M et al. Western Michigan Univ, Dept Comp Sci, Kalamazoo, MI 49008 USA.
ABSTRACT: The diversity of the available protein search engines with respect to the utilized matching algorithms, the low overlap ratios among their results and the disparity of their coverage encourage the community of proteomics to utilize ensemble solutions of different search engines. The advancing in cloud computing technology and the availability of distributed processing clusters can also provide support to this task. However, data transferring and results' combining, in this case, could be the major bottleneck.
[more...]
Use: pFind



Selective Enrichment and Quantification of N-Terminal Glycine Peptides via Sortase A Mediated Ligation
Analytical Chemistry. 2018. Cao, T et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG.
[more...]
Use: pFind



Myeloid-derived suppressor cells inhibit T cell activation through nitrating LCK in mouse cancers
Proceedings of the National Academy of Sciences of the United States of America. 2018. Feng, S et al. Univ Notre Dame, Dept Biol Sci, Notre Dame, IN 46556 USA.
ABSTRACT: Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs.
[more...]
Use: pFind



A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1$\alpha$ protein
PLOS Pathogens. 2018. Xu, CX et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan, Hubei, Peoples R China.
ABSTRACT: The essential role of pathogens in host metabolism is widely recognized, yet the mechanisms by which they affect host physiology remain to be fully defined. Here, we found that NIeB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to possess N-acetylglucosamine (GlcNAc) transferase activity, GlcNAcylates HIF-la, a master regulator of cellular O-2 homeostasis. We determined that NIeB-mediated GlcNAcylation at a conserved arginine 18 (Arg18) at the N-terminus of HIF-1 alpha enhanced HIF-1 alpha transcriptional activity, thereby inducing HIF-1 alpha downstream gene expression to alter host glucose metabolism.
[more...]
Use: pFind



Site-specific quantification of protein palmitoylation by cysteine-stable isotope metabolic labeling
Analytical Chemistry. 2018. Zhang, XQ et al. Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT: Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision.
[more...]
Use: pFind



A crude 1-DNJ extract from home made Bombyx batryticatus inhibits diabetic cardiomyopathy-associated fibrosis in db/db mice and reduces protein N-glycosylation levels
International Journal of Molecular Sciences. 2018. Zhao, Q et al. Liaoning Tradit Chinese Med Univ, Chinese Mat Med Proc Engn Technol Res Ctr Liaonin, Key Lab Chinese Mat Med Proc Principle Anal, State Adm Tradit Chinese Med,Pharmaceut Coll, Dalian 110060, Peoples R China.
ABSTRACT: The traditional Chinese drug Bombyx Batryticatus (BB), which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ) extracted from BB, we used the db/db (diabetic) mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS) identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins.
[more...]
Use: pFind



Enrichment-based proteogenomics identifies microproteins, missing proteins, and novel smORFs in Saccharomyces cerevisiae
Journal of Proteome Research. 2018. He, CT et al. Anhui Med Univ, Hefei 230032, Anhui, Peoples R China.
ABSTRACT: Microproteins are peptides composed of 100 amino acids (AA) or fewer, encoded by small open reading frames (smORFs). It has been demonstrated that microproteins participate in and regulate a wide range of functions in cells. However, the annotation and identification of microproteins is challenging in part owing to their low molecular weight, low abundancy, and hydrophobicity. These factors have led to the unannotation of smORFs in genome processing and have made their identification at the protein level difficult.
[more...]
Use: pFind



Synthesis, cytotoxic evaluation and target identification of thieno [2, 3-d] pyrimidine derivatives with a dithiocarbamate side chain at C2 position
EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY. 2018. Yang, CR et al. Capital Normal Univ, Dept Chem, Beijing 100048, Peoples R China.
ABSTRACT: Two series of thieno[2,3-d]pyrimidine derivatives bearing a dithiocarbamate side chain at the C2 position were synthesized and evaluated for cytotoxic activity in human lung cancer A549 and colon cancer HCT-116 cell lines. Compound 3n exhibited the most cytotoxic effect on A549 cells with an IC50 value of 4.87 mu M inducing a cell cycle arrest at G2/M phase and activating the spindle assembly checkpoint (SAC). To identify the target protein(s) of 3n, we incorporated biotin with 3n through a three-carbon chain and an amide bond to synthesize probe 10.
[more...]
Use: pFind



Large-scale differentiation and site specific discrimination of hydroxyproline isomers by electron transfer/higher-energy collision dissociation (EThcD) mass spectrometry
Analytical chemistry. 2018. Ma, Fengfei et al. Univ Wisconsin, Sch Pharm, Madison, WI 53705 USA; Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA
ABSTRACT: 3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples.
[more...]
Use: pGlyco; pFind



The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS
Electrophoresis. 2018. Yin, H et al. Hong Kong Polytech Univ, Shenzhen Res Inst, Shenzhen, Peoples R China.
ABSTRACT: A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures.
[more...]
Use: pFind; pGlyco



Long noncoding RNA AB074169 inhibits cell proliferation via modulation of KHSRP-mediated CDKN1a expression in papillary thyroid carcinoma
Cancer research. 2018. Gou, QH et al. Sichuan Univ, West China Hosp, Chengdu 610041, Sichuan, Peoples R China.
ABSTRACT: Long noncoding RNAs (lncRNA) are emerging as a novel class of regulators in gene expression associated with tumorigenesis. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) is poorly understood. Here, we conducted global lncRNA profiling and identified lncRNA AB074169 (lncAB) as significantly downregulated in PTC. Decreased expression of lncAB in PTC was caused by CpG hypermethylation within its gene promoter. Functional studies showed that lncAB overexpression led to cell-cycle arrest and tumor growth inhibition in vitro and in vivo, whereas lncAB knockdown promoted cell proliferation.
[more...]
Use: pLink



Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation
Journal of Biological Chemistry. 2018. Kaasboll, OJ et al. Oslo Univ Hosp, Inst Surg Res, A3-1057,POB 4950 Nydalen, NO-0424 Oslo, Norway.
ABSTRACT: Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved.
[more...]
Use: pLink



Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures
Biophysics Reports. 2018. Lu, Shan et al. Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), University of CAS, Institute of Computing Technology, CAS, Beijing 100190, China; National Institute of Biological Sciences, Beijing, Beijing 102206, China
ABSTRACT: Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography-mass spectrometry (LC-MS) and a powerful data analysis software tool named pLink.
[more...]
Use: pLink



Solution structure of extracellular loop of human $\beta$4 subunit of BK channel and its biological implication on ChTX sensitivity
Scientific Reports. 2018. Yanting Wang, Wenxian Lan et al. State Key Laboratory of Bioorganic and Natural Product Chemistry, Center for Excellence in Molecular Synthesis,Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences (CAS), 345 Lingling Road, Shanghai, 200032, China
ABSTRACT: Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming a subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BK alpha coexpression with auxiliary beta (beta 1-beta 4) subunits, as well as newly defined. subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched beta 4 subunit (h beta 4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. 
[more...]
Use: pLink



Modular organization and assembly of SWI/SNF family chromatin remodeling complexes
Cell. 2018. Mashtalir, N et al. Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02215 USA.
ABSTRACT: Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human ca; and developmental disorders. To date, the modular organization and path- ways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability.
[more...]
Use: pLink



Integrative structure and functional anatomy of a nuclear pore complex
NATURE. 2018. Kim, SJ et al. Univ Calif San Francisco, Dept Pharmaceut Chem, Dept Bioengn & Therapeut Sci, San Francisco, CA 94158 USA.
ABSTRACT: Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility.
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Structure and mechanogating mechanism of the Piezo1 channel
Nature. 2018. Zhao, QC et al. Tsinghua Univ, Sch Pharmaceut Sci Life Sci, Beijing 100084, Peoples R China.
ABSTRACT: The mechanosensitive Piezo channels function as key eukaryotic mechanotransducers. However, their structures and mechanogating mechanisms remain unknown. Here we determine the three-bladed, propeller-like electron cryo-microscopy structure of mouse Piezol and functionally reveal its mechanotransduction components. Despite the lack of sequence repetition, we identify nine repetitive units consisting of four transmembrane helices each-which we term transmembrane helical units (THUs) -which assemble into a highly curved blade-like structure.
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Structure of activated transcription complex Pol II–DSIF–PAF–SPT6
Nature. 2018. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Honto sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF.
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Structure of paused transcription complex Pol II–DSIF–NELF
Nature. 2018. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 angstrom resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate.
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Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose
Nature. 2018. Zhou, P et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-kappa B signalling(1). Recent studies indicate that the bacterial metabolite D-glycero-beta-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-kappa B signalling in host cytosol(2-4), but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-beta-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-kappa B activation and cytokine expression.
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Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity
Nature. 2018. Pao, KC et al. Univ Dundee, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland.
ABSTRACT: Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4).
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Structure and conformational dynamics of the human spliceosomal Bact complex
Cell. 2018. Haselbach, D et al. Max Planck Inst Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human B-act spliceosome at 3.4 angstrom resolution. In the B-act state, the spliceosome is activated but not catalytically primed, so that it is functionally blocked prior to the first catalytic step of splicing. The spliceosomal core is similar to the yeast B-act spliceosome; important differences include the presence of the RNA helicase aquarius and peptidyl prolyl isomerases.
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Structure of the core of the type III secretion system export apparatus
nature structural & molecular biology. 2018. Lucas Kuhlen , Patrizia Abrusci, Steven Johnson et al. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
ABSTRACT: Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 angstrom. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry.
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ROS-induced R loops trigger a transcription-coupled but BRCA1/2-independent homologous recombination pathway through CSB
NATURE COMMUNICATIONS. 2018. Teng, YQ et al. Univ Pittsburgh, Dept Microbiol & Mol Genet, Sch Med, 450 Technol Dr,523 Bridgeside Point 2, Pittsburgh, PA 15219 USA.
ABSTRACT: Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain.
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Structure of native lens connexin 46/50 intercellular channels by cryo-EM
Nature. 2018. Myers, JB et al. Portland State Univ, Dept Chem, Portland, OR 97207 USA.
ABSTRACT: Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50).
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Modular assembly of the nucleolar pre-60S ribosomal subunit
NATURE. 2018. Sanghai, ZA et al. Rockefeller Univ, Lab Prot & Nucle Acid Chem, New York, NY 10065 USA.
ABSTRACT: Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 angstrom.
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Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum
Nature Microbiology. 2018. Ozcan, A et al. Max Planck Inst Terr Microbiol, Marburg, Germany.
ABSTRACT: Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome.
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Cryo-EM structure of the exocyst complex
Nature Structural & Molecular Biology. 2018. Mei, KR et al. Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT: The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 angstrom. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core.
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Cryo-EM structure of human mTOR complex 2
Cell Research. 2018. Xizi Chen, Mengjie Liu et al. Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China;
ABSTRACT: Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 A resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry.
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Gcn4-mediator specificity is mediated by a large and dynamic fuzzy protein-protein complex
Cell Reports. 2018. Tuttle, LM et al. Fred Hutchinson Canc Res Ctr, Div Basic Sci, Seattle, WA 98109 USA.
ABSTRACT: Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions.
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Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1–Npl4
nature structural & molecular biology. 2018. Bodnar, NO et al. Harvard Med Sch, Howard Hughes Med Inst, Boston, MA 02115 USA.
ABSTRACT: Many polyubiquitinated proteins are extracted from membranes or complexes by the conserved ATPase Cdc48 (in yeast; p97 or VCP in mammals) before proteasomal degradation. Each Cdc48 hexamer contains two stacked ATPase rings (D1 and D2) and six N-terminal (N) domains. Cdc48 binds various cofactors, including the Ufd1-Npl4 heterodimer. Here, we report structures of the Cdc48-Ufd1-Npl4 complex from Chaetomium thermophilum. Npl4 interacts through its UBX-like domain with a Cdc48 N domain, and it uses two Zn2+-finger domains to anchor the enzymatically inactive Mpr1-Pad1 N-terminal (MPN) domain, homologous to domains found in several isopeptidases, to the top of the D1 ATPase ring.
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Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex
NATURE COMMUNICATIONS. 2018. Oroz, J et al. German Ctr Neurodegenerat Dis DZNE, Von Siebold Str 3a, D-37075 Gottingen, Germany.
ABSTRACT: The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity.
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Elongation/termination factor exchange mediated by PP1 phosphatase orchestrates transcription termination
Cell reports. 2018. Kecman, T et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5.
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The interaction network of the YidC insertase with the SecYEG translocon, SRP and the SRP receptor FtsY
Scientific reports. 2018. Petriman, NA et al. Albert Ludwigs Univ Freiburg, Fac Med, ZBMZ, Inst Biochem & Mol Biol, D-79104 Freiburg, Germany.
ABSTRACT: YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E.
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Carboxylate-selective chemical cross-linkers for mass spectrometric analysis of protein structures
Analytical chemistry. 2018. Zhang, XY et al. Peking Univ, Beijing Natl Lab Mol Sci,Minist Educ, Key Lab Bioorgan Chem & Mol Engn,Dept Chem Biol, Coll Chem & Mol Engn,Synthet & Funct Biomol Ctr, Beijing 100871, Peoples R China.
ABSTRACT: Chemical cross-linking coupled with mass spectrometry (CXMS) facilitates structural analysis, of proteins. As current CXMS applications are almost exclusively limitedto lysine residues, they Can only retrieve a small pOrtion of the structural information theoretically accessibleto CXMS. Chemical: cross-linkers targeting the acidic residues Asp/Glu could greatly enhance the power of CXMS. However, it has been difficult to develop chemistries that offer selectivity and efficiency under physiological conditions.
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TRiC controls transcription resumption after UV damage by regulating Cockayne syndrome protein A
Nature communications. 2018. Pines, A et al. Leiden Univ, Dept Human Genet, Med Ctr, Einthovenweg 20, NL-2333 ZC Leiden, Netherlands.
ABSTRACT: Transcription-blocking DNA lesions are removed by transcription-coupled nucleotide excision repair (TC-NER) to preserve cell viability. TC-NER is triggered by the stalling of RNA polymerase II at DNA lesions, leading to the recruitment of TC-NER-specific factors such as the CSA-DDB1-CUL4A-RBX1 cullin-RING ubiquitin ligase complex (CRLCSA). Despite its vital role in TC-NER, little is known about the regulation of the CRLCSA complex during TC-NER. Using conventional and cross-linking immunoprecipitations coupled to mass spectrometry, we uncover a stable interaction between CSA and the TRiC chaperonin.
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Prp19/Pso4 is an autoinhibited ubiquitin ligase activated by stepwise assembly of three splicing factors
Molecular Cell. 2018. de Moura, TR et al. Max Planck Inst Biophys Chem, Macromol Crystallog Grp, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis. Formation of the NTC core by stepwise assembly of SPF27, CDC5L, and PLRG1 onto the Prp19 tetramer enables ubiquitin ligation.
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Chemical crosslinking mass spectrometry reveals the conformational landscape of the activation helix of PPARγ; a model for ligand-dependent antagonism
STRUCTURE. 2018. Zheng, J et al. Scripps Res Inst, Dept Mol Med, Jupiter, FL 33458 USA.
ABSTRACT: Peroxisome proliferator-activated receptors (PPARs) are pharmacological targets for the treatment of metabolic disorders. Previously, we demonstrated the anti-diabetic effects of SR1664, a PPAR gamma modulator lacking classical transcriptional agonism, despite its poor pharmacokinetic properties. Here, we report identification of the antagonist SR11023 as a potent insulin sensitizer with significant plasma exposure following oral administration. To determine the structural mechanism of ligand-dependent antagonism of PPAR gamma, we employed an integrated approach combining solution-phase biophysical techniques to monitor activation helix (helix 12) conformational dynamics.
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Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping
nature protocols. 2018. Xiao, KH et al. Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15213 USA.
ABSTRACT: Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions.
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Molecular architecture of the essential yeast histone acetyltransferase complex NuA4 redefines its multimodularity
Molecular and Cellular Biology. 2018. Setiaputra, D et al. Univ British Columbia, Dept Biochem & Mol Biol, Vancouver, BC, Canada.
ABSTRACT: Conserved from yeast to humans, the NuA4 histone acetyltransferase is a large multisubunit complex essential for cell viability through the regulation of gene expression, genome maintenance, metabolism, and cell fate during development and stress. How the different NuA4 subunits work in concert with one another to perform these diverse functions remains unclear, and addressing this central question requires a comprehensive understanding of NuA4's molecular architecture and subunit organization. We have determined the structure of fully assembled native yeast NuA4 by single-particle electron microscopy.
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Development of in Planta Chemical Cross-Linking-Based Quantitative Interactomics in Arabidopsis
Journal of proteome research. 2018. Liu, SC et al. Hong Kong Univ Sci & Technol, Inst Environm, Energy Inst, Div Life Sci, Hong Kong, Peoples R China.
ABSTRACT: An in planta chemical cross-linking-based quantitative interactomics (IPQCX-MS) workflow has been developed to investigate in vivo protein protein interactions and alteration in protein structures in a model organism, Arabidopsis thaliana. A chemical cross-linker, azide-tag-modified disuccinimidyl pimelate (AMDSP), was directly applied onto Arabidopsis tissues. Peptides produced from protein fractions of CsCl density gradient centrifugation were dimethyl-labeled, from which the AMDSP cross-linked peptides were fractionated on chromatography, enriched, and analyzed by mass spectrometry.
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Architecture and subunit arrangement of the complete Saccharomyces cerevisiae COMPASS complex
Scientific Reports. 2018. Wang, YX et al. Univ Chinese Acad Sci, Chinese Acad Sci, Natl Ctr Prot Sci Shanghai,State Key Lab Mol Biol, CAS Ctr Excellence Mol Cell Sci,Shanghai Inst Bio, Shanghai 201210, Peoples R China.
ABSTRACT: Methylation of histone H3 lysine 4 (H3K4) is catalyzed by the multi-component COMPASS or COMPASS-like complex, which is highly conserved from yeast to human, and plays essential roles in gene expression and transcription, cell cycle progression, and DNA repair. Here we present a cryo-EM map of the complete S. cerevisiae COMPASS complex. Through tag or Fab labeling strategy combined with cryo-EM 3D reconstruction and cross-linking and mass spectrometry (XL-MS) analysis, we uncovered new information on the subunit arrangement: Cps50, Cps35, and Cps30 were determined to group together to form the face region in the head of the complex, and Cps40 and the N-terminal portion of Set1 reside on the top of the head.
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Oligomerisation of Synaptobrevin-2 studied by native mass spectrometry and chemical cross-linking
Journal of the American Society for Mass Spectrometry. 2018. Wittig, S et al. Martin Luther Univ Halle Wittenberg, Inst Biochem & Biotechnol, Charles Tanford Prot Ctr, Interdisciplinary Res Ctr HALOmem, Kurt Mothes Str 3a, D-06120 Halle, Saale, Germany.
ABSTRACT: Synaptobrevin-2 is a key player in signal transmission in neurons. It forms, together with SNAP25 and Syntaxin-1A, the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and mediates exocytosis of synaptic vesicles with the pre-synaptic membrane. While Synaptobrevin-2 is part of a four-helix bundle in this SNARE complex, it is natively unstructured in the absence of lipids or other SNARE proteins. Partially folded segments, presumably SNARE complex formation intermediates, as well as formation of Synaptobrevin-2 dimers and oligomers, were identified in previous studies.
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Identification of a novel tetrameric structure for human apolipoprotein-D
Journal of Structural Biology. 2018. Kielkopf, CS et al. Univ Wollongong, Illawarra Hlth & Med Res Inst, Room 230,Bldg 32,Northfields Ave, Wollongong, NSW 2522, Australia.
ABSTRACT: Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded beta-barrel with a single adjacent a-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-beta burden in Alzheimer's disease mouse models.
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Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation
Journal of Biological Chemistry. 2018. Sugita, S et al. Konan Univ, Fac Sci & Engn, Dept Biol, Okamoto 8-9-1, Kobe, Hyogo 6588501, Japan.
ABSTRACT: ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA(+)), ClpB forms a hexameric ring structure, with each protomer containing two AAA(+) modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring.
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Structural basis for the regulatory interaction of the methylglyoxal synthase MgsA with the carbon flux regulator Crh in Bacillus subtilis
Journal of Biological Chemistry. 2018. Dickmanns, A et al. Georg August Univ Gottingen, Dept Mol Struct Biol, Justus von Liebig Weg 11, D-37077 Gottingen, Germany.
ABSTRACT: Utilization of energy-rich carbon sources such as glucose is fundamental to the evolutionary success of bacteria. Glucose can be catabolized via glycolysis for feeding the intermediary metabolism. The methylglyoxal synthase MgsA produces methylglyoxal from the glycolytic intermediate dihydroxyacetone phosphate. Methylglyoxal is toxic, requiring stringent regulation of MgsA activity. In the Gram-positive bacterium Bacillus subtilis, an interaction with the phosphoprotein Crh controls MgsA activity.
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Modification of the reaction system of Ara h 2 catalyzed by MTGase: Products and reaction conditions analysis
Journal of Food Processing and Preservation. 2018. Wu, ZH et al. Nanchang Univ, State Key Lab Food Sci & Technol, Nanchang 330047, Jiangxi, Peoples R China.
ABSTRACT: Peanut (Arachis hypogaea) is listed among the eight major food allergens, in which Ara h 2 is the major allergen. The microbial transglutaminase (MTGase)-catalyzed cross-linking reaction reduces the allergenicity of Ara h 2. However, deamidation might occur and influence the cross-linking reaction. In this work, native and reduced Ara h 2 were catalyzed by MTGase. In addition to intermolecular cross-linking, intramolecular cross-linking and deamidation were proven to occur simultaneously. Moreover, intramolecular cross-linking sites were identified using mass spectrometry and the PLINK software.
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Molecular structure of promoter-bound yeast TFIID
Nature Communications. 2018. Kolesnikova, O et al. Inst Genet & Biol Mol & Cellulaire, Equipe Labellisee Ligue Canc, Dept Integrated Struct Biol, F-67404 Illkirch Graffenstaden, France.
ABSTRACT: Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 A, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID.
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Proximity-enhanced SuFEx chemical cross-linker for specific and multitargeting cross-linking mass spectrometry
PNAS. 2018. Yang, B et al. Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94158 USA.
ABSTRACT: Chemical cross-linking mass spectrometry (CXMS) is being increasingly used to study protein assemblies and complex protein interaction networks. Existing CXMS chemical cross-linkers target only Lys, Cys, Glu, and Asp residues, limiting the information measurable. Here we report a "plant-and-cast" cross-linking strategy that employs a heterobifunctional cross-linker that contains a highly reactive succinimide ester as well as a less reactive sulfonyl fluoride. The succinimide ester reacts rapidly with surface Lys residues "planting" the reagent at fixed locations on protein.
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Structure of activated transcription complex Pol II--DSIF--PAF--SPT6
Nature. 2018. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT: Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Honto sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF.
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EpiProfile 2.0: a computational platform for processing epi-proteomics mass spectrometry data
Journal of Proteome Research. 2018. Yuan, ZF et al. Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Inst, Philadelphia, PA 19104 USA.
ABSTRACT: Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
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Peptide Sequencing in Graphs for Multiplex Mass Spectra
IEEE Latin America Transactions. 2018. Damaso, JCG et al. Univ Fed Rio de Janeiro, Rio De Janeiro, Brazil.
ABSTRACT: This work presents a heuristic to the peptide sequencing in multiplex mass spectra. We developed a methodology that deconvolutes the multiplex mass spectrum, generating a modified specific spectrum for each peptide target mass. A directed spectrum graph is created from each deconvoluted spectrum and a cost function is defined to guide the depth first search algorithm through the sequence paths. The heuristic considers that the peptide with higher intensity would be correctly sequenced from the intact multiplex spectrum.
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CharmeRT: boosting peptide identifications by chimeric spectra identification and retention time prediction
Journal of proteome research. 2018. Dorfer, V et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, Softwarepk 11, A-4232 Hagenberg, Austria.
ABSTRACT: Coeluting peptides are still a major challenge for the identification and validation of MS/MS spectra, but carry great potential. To tackle these problems, we have developed the here presented CharmeRT workflow, combining a chimeric spectra identification strategy implemented as part of the MS Amanda algorithm with the validation system Elutator, which incorporates a highly accurate retention time prediction algorithm. For high resolution data sets this workflow identifies 38-64% chimeric spectra, which results in up to 63% more unique peptides compared to a conventional single search strategy.
Use: pParse



CharmeRT: Boosting peptide identifications by chimeric spectra identification and retention time prediction
Journal of proteome research. 2018. Dorfer, V et al. Univ Appl Sci Upper Austria, Bioinformat Res Grp, Softwarepk 11, A-4232 Hagenberg, Austria.
ABSTRACT: Coeluting peptides are still a major challenge for the identification and validation of MS/MS spectra, but carry great potential. To tackle these problems, we have developed the here presented CharmeRT workflow, combining a chimeric spectra identification strategy implemented as part of the MS Amanda algorithm with the validation system Elutator, which incorporates a highly accurate retention time prediction algorithm. For high resolution data sets this workflow identifies 38-64% chimeric spectra, which results in up to 63% more unique peptides compared to a conventional single search strategy.
Use: pParse



Joint precursor elution profile inference via regression for peptide detection in data-independent acquisition mass spectra
Journal of proteome research. 2018. Hu, A et al. Box 355065,Foege Bldg,S220B,3720 15th Ave NE, Seattle, WA 98195 USA.
ABSTRACT: In data independent acquisition (DIA) mass spectrometry, precursor scans are interleaved with wide-window fragmentation scans, resulting in complex fragmentation spectra containing multiple coeluting peptide species. In this setting, detecting the isotope distribution profiles of intact peptides in the precursor scans can be a critical initial step in accurate peptide detection and quantification. This peak detection step is particularly challenging when the isotope peaks associated with two different peptide species overlap-or interfere-with one another.
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Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation
Chemical Science. 2018. Park, DD et al. Univ Calif Davis, Dept Chem, Davis, CA 95616 USA.
ABSTRACT: Given that unnatural sugar expression is metabolically achieved, the kinetics and disposition of incorporation can lend insight into the temporal and localization preferences of sialylation across the cell surface. However, common detection schemes lack the ability to detail the molecular diversity and distribution of target moieties. Here we employed a mass spectrometric approach to trace the placement of azido sialic acids on membrane glycoconjugates, which revealed substantial variations in incorporation efficiencies between N-/O-glycans, glycosites, and glycosphingolipids.
[more...]
Use: pGlyco



Study on behaviors and performances of universal N-glycopeptide enrichment methods
Analyst. 2018. Xue, Y et al. Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT: Glycosylation is a crucial process in protein biosynthesis. However, the analysis of glycopeptides through MS remains challenging due to the microheterogeneity and macroheterogeneity of the glycoprotein. Selective enrichment of glycopeptides from complex samples prior to MS analysis is essential for successful glycoproteome research. In this work, we systematically investigated the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC of glycopeptide enrichment to promote understanding of these methods.
[more...]
Use: pGlyco



Site-specific N-glycan characterization of grass carp serum IgM
Frontiers in Immunology. 2018. Su, YL et al. Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan, Hubei, Peoples R China.
ABSTRACT: Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied.
[more...]
Use: pGlyco



Phenylboronic acid functionalized C3N4 facultative hydrophilic materials for enhanced enrichment of glycopeptides
Talanta. 2018. Zhang, Y et al. Natl Ctr Prot Sci Beijing, Beijing Inst Life, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: It is challenging to capture N-glycopeptides with high recovery and high specificity from complicated biosystems. Herein, we present a facile and economical procedure to generate a novel self-assembling 4-Mercaptobenzene boronic acid functionalized and Au-doped Straticulate C3N4 (MASC), with enhanced affinity capability towards glycopeptides. The materials possess low pH value adaptation, high hydrophilicity and stability, good repeatability and recyclability, and provided high selectivity (1:100), low limit of detection (0.33 fmol/mu L), high enrichment efficiency (similar to 80%) and high recovery rate (similar to 90%) towards glycopeptides.
[more...]
Use: pGlyco



Quantitative analysis of newly synthesized proteins
Nature Protocol. 2018. Yuanhui Ma et al. Departments of Molecular Medicine and Neurobiology, The Scripps Research Institute, La Jolla, CA, USA.
ABSTRACT: Measuring proteome response to perturbations is critical for understanding the underlying mechanisms involved. Traditional quantitative proteomic methods are limited by the large numbers of proteins in the proteome and the mass spectrometer’s dynamic range. A previous method uses the biorthogonal reagent azidohomoalanine (AHA), a methionine analog, for labeling, enrichment and detection of newly synthesized proteins (NSPs). Newly synthesized AHA proteins can be coupled to biotin via CuAAC-mediated click chemistry and enriched using avidin-based affinity purification.
[more...]
Use: pQuant



Quantitative temporal analysis of protein dynamics in cardiac remodeling
Journal of molecular and cellular cardiology. 2018. McClatchy, DB et al. Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT: Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle.
[more...]
Use: pQuant



Quantitative Temporal Analysis of Protein Dynamics in Maladaptive Cardiac Remodeling
Journal of molecular and cellular cardiology. 2018. McClatchy, DB et al. Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.
ABSTRACT: Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle.
[more...]
Use: pQuant




2017




Different proteome profiles between male and female Populus cathayana exposed to UV-B radiation
Frontiers in plant science. 2017. Zhang, Yunxiang et al. Chinese Acad Sci, Inst Mt Hazards & Environm, Key Lab Mt Surface Proc & Ecol Regulat, Chengdu, Peoples R China
ABSTRACT: With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear.
[more...]
Use: pFind



In-depth proteome coverage by improving efficiency for membrane proteome analysis
Analytical chemistry. 2017. Zhao, Qun et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: Although great achievement has been made in the mapping of human proteome, the efficiency of sample preparation still needs to be improved, especially for membrane proteins. Herein, we presented a novel method to deepen proteome coverage by the sequential extraction of proteins using urea and 1-dodecy1-3- methylimidazolium chloride (C12Im-Cl). With such a strategy, the commonly lost hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl, as well as the suppression effect of high abundance soluble proteins could be decreased.
[more...]
Use: pFind



O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress
Nature Communications. 2017. Han, CF et al. Ocean Univ China, Sch Med & Pharm, 5 Yushan Rd, Qingdao 266003, Peoples R China.
ABSTRACT: SIRT1 is the most evolutionarily conserved mammalian sirtuin, and it plays a vital role in the regulation of metabolism, stress responses, genome stability, and ageing. As a stress sensor, SIRT1 deacetylase activity is significantly increased during stresses, but the molecular mechanisms are not yet fully clear. Here, we show that SIRT1 is dynamically modified with O-GlcNAc at Ser 549 in its carboxy-terminal region, which directly increases its deacetylase activity both in vitro and in vivo. The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative, and metabolic stress stimuli in cellular and mouse models, thereby increasing SIRT1 deacetylase activity and protecting cells from stress-induced apoptosis.
[more...]
Use: pFind



Proteomic analysis of exported chaperone/co-chaperone complexes of P. falciparum reveals an array of complex protein-protein interactions
Scientific reports. 2017. Zhang, Qi et al. Philipps Univ Marburg, Dept Parasitol, Marburg, Germany
ABSTRACT: Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure.
[more...]
Use: pFind



The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage
Molecular Biosystems. 2017. Karch, KR et al. Univ Penn, Epigenet Inst, Perelman Sch Med, Dept Biochem & Mol Biophys, Philadelphia, PA 19104 USA.
ABSTRACT: ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is critical in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive analysis of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by dimethyl sulfate (DMS).
[more...]
Use: pFind



Multi-protease strategy identifies three PE2 missing proteins in human testis tissue
Journal of Proteome Research. 2017. Wang, YH et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Although 5 years of the missing proteins (MPs) study have been completed, searching for MPs remains one of the core missions of the Chromosome-Centric Human Proteome Project (C-HPP). Following the next-50-MPs challenge of the C-HPP, we have focused on the testis-enriched MPs by various strategies since 2015. On the basis of the theoretical analysis of MPs (2017-01, neXtProt) using multiprotease digestion, we found that nonconventional proteases (e.g. LysargiNase, GluC) could improve the peptide diversity and sequence coverage compared with Trypsin.
[more...]
Use: pFind; pBuild; pLabel



Identification of missing proteins in the phosphoproteome of kidney cancer
Journal of Proteome Research. 2017. Peng, XH et al. Wuhan Univ, Sch Pharmaceut Sci, Minist Educ, Key Lab Combinatorial Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.
ABSTRACT: Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search biochemical strategies. MPs mainly fall into the following adequate and accurate evidence of MPs through various classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid associated proteins, (5) low abundance, and (6) unexpressed genes.
[more...]
Use: pFind; pBuild



Exhaustively identifying cross-linked peptides with a linear computational complexity
Journal of Proteome Research. 2017. Yu, FC et al. Hong Kong Univ Sci & Technol, Dept Elect & Comp Engn, Hong Kong, Hong Kong, Peoples R China.
ABSTRACT: Chemical cross-linking coupled to mass spectrometry is a powerful tool to study protein protein interactions and protein conformations. Two linked peptides are ionized and fragmented to produce a tandem mass spectrum. In such an experiment, a tandem mass spectrum contains ions from two peptides. The peptide identification problem becomes a peptide peptide pair identification problem. Currently, most tools do not search all possible pairs due to the quadratic time complexity. Consequently, missed findings are unavoidable.
[more...]
Use: pLink; pFind



An insight into glyco-microheterogeneity of plasma von Willebrand factor by mass spectrometry
Journal of Proteome Research. 2017. Gashash, EA et al. Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30302 USA.
ABSTRACT: Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms.
[more...]
Use: pFind



Different Proteome Profiles between Male and Female Populus cathayana Exposed to UV-B Radiation
Frontiers in plant science. 2017. Zhang, Yunxiang et al. Chinese Acad Sci, Inst Mt Hazards & Environm, Key Lab Mt Surface Proc & Ecol Regulat, Chengdu, Peoples R China
ABSTRACT: With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear.
[more...]
Use: pFind



Proteomics investigations into serum proteins adsorbed by high‐flux and low‐flux dialysis membranes
Proteomics Clinical Applications. 2017. Han, S et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian, Peoples R China.
ABSTRACT: Hemodialysis is one of the most important therapies for patients with uremia, and the dialysis membrane is the predominant factor that impacts the efficiency of dialysis. Here, a protein adsorption on two different membranes is investigated to provide a basis for improving dialysis materials. Two cases treated with the Polyflux 14L low-flux dialyzer and the Polyflux 140H high-flux dialyzers during two continuous therapies are selected. Four used dialyzers from selected patients are infused with C12Im-Cl to elute the adsorbed proteins.
[more...]
Use: pFind



A rapid and easy protein N‐terminal profiling strategy using (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) labeling …
Proteomics. 2017. Li, YC et al. Beijing Proteome Res Ctr, 38 Sci Pk Rd, Beijing 102206, Peoples R China.
ABSTRACT: Protein N-terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N-terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N-terminal peptide losses. In this study, a fast, one-step approach utilizing (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N-terminal peptide enrichment and analysis.
[more...]
Use: pFind



Protein-level integration strategy of multiengine MS spectra search results for higher confidence and sequence coverage
Journal of Proteome Research. 2017. Zhao, PP et al. Jinan Univ, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Inst Life & Hlth Engn, Coll Life Sci & Technol, Guangzhou 510632, Guangdong, Peoples R China.
ABSTRACT: Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines.
[more...]
Use: pFind



Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides
Analytical and Bioanalytical Chemistry. 2017. Yu, ZX et al. Anhui Med Univ, 81 Meishan Rd, Hefei 230032, Anhui, Peoples R China.
ABSTRACT: Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans.
[more...]
Use: pFind; pGlyco



Chemoproteomics reveals unexpected lysine/arginine-specific cleavage of peptide chains as a potential protein degradation machinery
Analytical Chemistry. 2017. Tian, CP et al. Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Proteins can undergo oxidative cleavage by in-vitro metal-catalyzed oxidation (MCO) in either the aamidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal alpha-ketoacyl products and >1000 unexpected N-terminal formyl products.
[more...]
Use: pFind; pQuant



A chemoproteomic platform to assess bioactivation potential of drugs
Chemical Research in Toxicology. 2017. Sun, R et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, Natl Ctr Prot Sci, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Reactive metabolites (RM) formed from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of major cytochrome P450 (CYP450) enzymes. Risk of bioactivation of a test compound is routinely examined as part Of lead optimization efforts in drug discovery. Here we described a chemoproteomic platform to ass in vitro and in vivo bioactivation potential of drugs. This platform enabled us to determine reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes and living animals.
[more...]
Use: pFind; pQuant



The Null-Test for peptide identification algorithm in Shotgun proteomics
Journal of Proteomics. 2017. Zhang, SR et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian, Peoples R China.
ABSTRACT: The present research proposed general evaluation strategy named Null-Test for peptide identification algorithm in Shotgun proteomics. The Null-Test method based on random matching can be utilized to check whether the algorithm has a tendency to make a mistake or has potential bugs, faultiness, errors etc., and to validate the reliability of the identification algorithm. Unfortunately, none of the five famous identification software could pass the most stringent Null-Test. PatternLab had good performance in both Null-Test and routine search by making a good control on the overfitting with sound design.
[more...]
Use: pFind



Towards Centralized MS/MS Spectra Preprocessing: An Empirical Evaluation of Peptides Search Engines using Ground Truth Datasets
2017 IEEE 17TH INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOENGINEERING (BIBE). 2017. Maabreh, Majdi et al. Western Michigan Univ, Dept Comp Sci, Kalamazoo, MI 49008 USA
ABSTRACT: several peptides search engines have been developed in the recent decades. Most of the time and for the same inputs, different search engines' result in different peptides were identified, which can confuse the stakeholders in the field of proteomics. The massive amount of generated spectra by high throughput spectrometers adds another challenge which handicaps the current search engines. This motivates the researchers to evaluate the combination of several search engines. Several studies provided ensemble solutions over shared and distributed computing environments for reliable results.
[more...]
Use: pFind



Deep vs. shallow learning-based filters of MS/MS spectra in support of protein search engines
2017 IEEE INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOMEDICINE (BIBM). 2017. Maabreh, Majdi et al. Western Michigan Univ, Dept Comp Sci, Kalamazoo, MI 49008 USA
ABSTRACT: Despite the linear relation between the number of observed spectra and the searching time, the current protein search engines, even the parallel versions, could take several hours to search a large amount of MS/MS spectra, which can be generated in a short time. After a laborious searching process, some (and at times, majority) of the observed spectra are labeled as non-identifiable. We evaluate the role of machine learning in building an efficient MS/MS filter to remove non-identifiable spectra.
[more...]
Use: pFind



Different proteome profiles between male and female Populus cathayana exposed to UV-B radiation
Frontiers in plant science. 2017. Zhang, Yunxiang et al. Chinese Acad Sci, Inst Mt Hazards & Environm, Key Lab Mt Surface Proc & Ecol Regulat, Chengdu, Peoples R China
ABSTRACT: With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear.
[more...]
Use: pFind



Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery
Analytical Chemistry. 2017. Tian, CP et al. Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Proteins can undergo oxidative cleavage by in-vitro metal-catalyzed oxidation (MCO) in either the aamidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal alpha-ketoacyl products and >1000 unexpected N-terminal formyl products.
[more...]
Use: pFind; pQuant



Proteomic study provides new clues for complications of hemodialysis caused by dialysis membrane
Science Bulletin. 2017. Yang, KG et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT: The complications of hemodialysis accompanied the hemodialysis and threaten the patients' life. Besides the loss of nutrient substance, such as amino acid and vitamin, we found new clues that the adsorbed proteins on common-used polysulfone-based dialysis membrane might be the reason according to the qualitative proteomic study by ionic liquid assisted sample preparation method. Our results indicated that the adsorbed proteins on the membrane were related with complement activation, blood coagulation, and leukocyte-related biological process.
[more...]
Use: pFind



A rapid and easy protein N-terminal profiling strategy using (N-Succinimidyloxycarbonylmethyl) tris (2, 4, 6-trimethoxyphenyl) phosphonium bromide (TMPP) labeling and StageTip
Proteomics. 2017. Li, YC et al. Beijing Proteome Res Ctr, 38 Sci Pk Rd, Beijing 102206, Peoples R China.
ABSTRACT: Protein N-terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N-terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N-terminal peptide losses. In this study, a fast, one-step approach utilizing (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N-terminal peptide enrichment and analysis.
[more...]
Use: pFind



Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers
Science Translational Medicine. 2017. Kim, J et al. Univ Penn, Inst Regenerat Med, Dept Cell & Dev Biol, Abramson Canc Ctr,Tumor Biol Program,Perelman Sch, 9-131 Smilow Ctr Translat Res,3400 Civ Ctr Blvd, Philadelphia, PA 19104 USA.
ABSTRACT: Markers are needed to facilitate early detection of pancreatic ductal adenocarcinoma (PDAC), which is often diagnosed too late for effective therapy. Starting with a PDAC cell reprogramming model that recapitulated the progression of human PDAC, we identified secreted proteins and tested a subset as potential markers of PDAC. We optimized an enzyme-linked immunosorbent assay (ELISA) using plasma samples from patients with various stages of PDAC, from individuals with benign pancreatic disease, and from healthy controls.
[more...]
Use: pFind



Aptamer-immobilized open tubular capillary column to capture circulating tumor cells for proteome analysis
Talanta. 2017. Liu, LK et al. 457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT: Circulating tumor cells hold the key to predicting the prognosis and discovering the therapeutic targets. Herein, we proposed a strategy to develop an aptamer-immobilized open tubular capillary column by which SMMC-7721 human hepatoma cells (SMMC-7721 cells) could be captured with an over 70% of capture efficiency and a 3.0 +/- 0.2 of enrichment factor. Owing to the compatibility of the column, the captured cells by the column could be analyzed by LC-MS from protein level and 5 unique proteins of SMMC-7721 cells were identified which could be used as markers to identify SMMC-7721 cells when Jurkat T-leukemia cells (Jurkat cells) were employed as interfering cells.
[more...]
Use: pFind



Proteomics Investigations into Serum Proteins Adsorbed by High-Flux and Low-Flux Dialysis Membranes
Proteomics Clinical Applications. 2017. Han, S et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian, Peoples R China.
ABSTRACT: Hemodialysis is one of the most important therapies for patients with uremia, and the dialysis membrane is the predominant factor that impacts the efficiency of dialysis. Here, a protein adsorption on two different membranes is investigated to provide a basis for improving dialysis materials. Two cases treated with the Polyflux 14L low-flux dialyzer and the Polyflux 140H high-flux dialyzers during two continuous therapies are selected. Four used dialyzers from selected patients are infused with C12Im-Cl to elute the adsorbed proteins.
[more...]
Use: pFind



基于梯度提升决策树的肽碎片离子强度建模
山东理工大学学报(自然科学版). 2017. 怀 浩; 刘 学; 张龙波; 王晓丹 et al. 山东理工大学 计算机科学与技术学院 ,山东 淄博 255049
ABSTRACT: 为找到对蛋白质鉴定算法影响较大的肽碎片离子特征,以提高鉴定结果的正确率,在碎片离子特征与强度信息的基础上进行建模,构建预测模型.实验首先使用pFind对串联质谱数据鉴定,将鉴定结果过滤出需要的肽序列;然后计算出离子质荷比与离子特征值,通过匹配离子的质荷比获取离子强度信息;使用强度信息与离子特征值构建libsvm格式文件,使用XGBoost构建预测模型,其中使用了梯度提升决策树算法;最后使用构建完成的预测模型对蛋白质产生的肽序列做离子强度理论预测.实验结果表明模型所预测的肽序列离子强度与实验离子强度有着较高的相似度,同时分析预测模型可以从预测树中发现肽序列碎裂的规律,提取肽碎片离子中对强度值影响较大的离子特征.
Use: pFind



Hepatitis B virus X protein stimulates proliferation, wound closure and inhibits apoptosis of HuH-7 cells via CDC42
International Journal of Molecular Sciences. 2017. Xu, YR et al. Natl Ctr Prot Sci Beijing, Natl Engn Res Ctr Prot Drugs, State Key Lab Prote, Beijing Proteome Res Ctr,Inst Radiat Med, Beijing 102206, Peoples R China.
ABSTRACT: Chronic hepatitis B virus (HBV) infection has been considered as the major cause of hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) has been reported to be oncogenic. The underlying mechanisms of HBV-related HCC are not fully understood, and the role played by the HBx protein in HBV induced carcinogenesis remains controversial. CDC42, a member of the Rho GTPase family, has been reported to be overexpressed in several different cancers, including HBV-related HCC. However, the specific role of CDC42 in HCC development remains unclear.
[more...]
Use: pFind



Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope
PNAS. 2017. Wu, Y et al. Chinese Acad Sci, Key Lab Pathogen Microbiol & Immunol, Inst Microbiol, Beijing 100101, Peoples R China.
ABSTRACT: Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements.
[more...]
Use: pLink; pParse



The SAMHD1 dNTP triphosphohydrolase is controlled by a redox switch
Antioxidants & Redox Signaling. 2017. Mauney, CH et al. Wake Forest Sch Med, Ctr Struct Biol, Dept Biochem, Winston Salem, NC 27157 USA.
ABSTRACT: Aims: Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Results: Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity.
[more...]
Use: pLink



Complete mapping of complex disulfide patterns with closely-spaced cysteines by in-source reduction and data-dependent mass spectrometry
Analytical chemistry. 2017. Cramer, CN et al. Novo Nordisk AS, Global Res, Prot Engn, Novo Nordisk Pk, DK-2760 Malov, Denmark.
ABSTRACT: Mapping of disulfide bonds is, an essential part of protein characterization to ensure correct cysteine pairings. For this, mass spectrometry (MS) is the most widely used technique due to fast and accurate Characterization. However, MS-based disulfide mapping is challenged when multiple disulfide bonds are present in complicated patterns. This includes the presence of disulfide bonds in nested patterns and closely spaced cysteines. Unambiguous mapping of such disulfide bonds typically requires advanced MS approaches.
[more...]
Use: pLink



DISC: DISulfide linkage Characterization from tandem mass spectra
Bioinformatics. 2017. Liu, Y et al. Univ Western Ontario, Dept Comp Sci, London, ON N6A 5B7, Canada.
ABSTRACT: Motivation: Enzymatic digestion under appropriate reducing conditions followed by mass spectrometry analysis has emerged as the primary method for disulfide bond analysis. The large amount of mass spectral data collected in the mass spectrometry experiment requires effective computational approaches to automate the interpretation process. Although different approaches have been developed for such purpose, they always choose to ignore the frequently observed internal ion fragments and they lack a reasonable quality control strategy and calibrated scoring scheme for the statistical validation and ranking of the reported results.
[more...]
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Structures of transcription pre-initiation complex with TFIIH and Mediator
Nature. 2017. Schilbach, S et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7 angstrom and 5.8 angstrom, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively.
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Cryo-EM structure of a human spliceosome activated for step 2 of splicing
Nature. 2017. Bertram, K et al. MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 angstrom, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains.
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Structure of the human MHC-I peptide-loading complex
NATURE. 2017. Blees, A et al. Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Strasse 9, Frankfurt/Main, 60438, Germany
ABSTRACT: The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide–MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells1,2.
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Cryo-EM structure of a pre-catalytic human spliceosome primed for activation
Cell. 2017. Bertram, K et al. MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6. U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step.
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Molecular architecture of the 90S small subunit pre-ribosome
eLife. 2017. Sun, Q et al. Chinese Acad Sci, CAS Ctr Excellence Biomacromol, Inst Biophys, Key Lab RNA Biol, Beijing, Peoples R China.
ABSTRACT: Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors.
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The complete structure of the small-subunit processome
nature structural & molecular biology. 2017. Barandun, J et al. Rockefeller Univ, Lab Prot & Nucle Acid Chem, 1230 York Ave, New York, NY 10021 USA.
ABSTRACT: The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 angstrom, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1.
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Structure of an intron lariat spliceosome from Saccharomyces cerevisiae
Cell. 2017. Wan, RX et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing 100084, Peoples R China.
ABSTRACT: The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 angstrom. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 50 exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly.
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Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation
Autophagy. 2017. Zheng, JX et al. Tsinghua Univ, Tsinghua Univ Peking Univ Joint Ctr Life Sci, Sch Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China.
ABSTRACT: PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs.
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Mechanism of transcription anti-termination in human mitochondria
Cell. 2017. Hillen, HS et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC).
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Structural insights into POT1-TPP1 interaction and POT1 C-terminal mutations in human cancer
Nature communications. 2017. Chen, C et al. Yale Sch Med, Dept Lab Med, New Haven, CT 05620 USA.
ABSTRACT: Mammalian shelterin proteins POT1 and TPP1 form a stable heterodimer that protects chromosome ends and regulates telomerase-mediated telomere extension. However, how POT1 interacts with TPP1 remains unknown. Here we present the crystal structure of the C-terminal portion of human POT1 (POT1C) complexed with the POT1-binding motif of TPP1. The structure shows that POT1C contains two domains, a third OB fold and a Holliday junction resolvase-like domain. Both domains are essential for binding to TPP1.
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Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions
Nature communications. 2017. Yang, B et al. Univ Calif San Francisco, Dept Pharmaceut Chem, 555 Mission Bay Blvd, San Francisco, CA 94158 USA.
ABSTRACT: Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells.
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Exploitation of an iron transporter for bacterial protein antibiotic import
PNAS. 2017. White, P et al. Univ Oxford, Dept Biochem, Oxford OX1 3QU, England.
ABSTRACT: Unlike their descendants, mitochondria and plastids, bacteria do not have dedicated protein import systems. However, paradoxically, import of protein bacteriocins, the mechanisms of which are poorly understood, underpins competition among pathogenic and commensal bacteria alike. Here, using X-ray crystallography, isothermal titration calorimetry, confocal fluorescence microscopy, and in vivo photoactivatable cross-linking of stalled translocation intermediates, we demonstrate how the iron transporter FpvAI in the opportunistic pathogen Pseudomonas aeruginosa is hijacked to translocate the bacteriocin pyocin S2 (pyoS2) across the outer membrane (OM).
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Loss of Snf5 induces formation of an aberrant SWI/SNF complex
Cell reports. 2017. Sen, P et al. UT MD Anderson Canc Ctr, Smithville, TX 78597 USA.
ABSTRACT: The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14.
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Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex
Nature Communications. 2017. Xu, YW et al. Max Planck Gesell, Max Planck Inst Biophys Chem, Dept Mol Biol, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: The conserved polymerase-associated factor 1 complex (Paf1C) plays multiple roles in chromatin transcription and genomic regulation. Paf1C comprises the five subunits Paf1, Leo1, Ctr9, Cdc73 and Rtf1, and binds to the RNA polymerase II (Pol II) transcription elongation complex (EC). Here we report the reconstitution of Paf1C from Saccharomyces cerevisiae, and a structural analysis of Paf1C bound to a Pol II EC containing the elongation factor TFIIS. Cryo-electron microscopy and crosslinking data reveal that Paf1C is highly mobile and extends over the outer Pol II surface from the Rpb2 to the Rpb3 subunit.
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Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa
Nucleic Acids Research. 2017. Sonnleitner, E et al. Univ Vienna, Dept Microbiol Immunobiol & Genet, Max F Perutz Labs, Vienna Bioctr, Dr Bohrgasse 9, A-1030 Vienna, Austria.
ABSTRACT: In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA.
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Structural basis for λN-dependent processive transcription antitermination
Nature Microbiology. 2017. Said, N et al. Free Univ Berlin, Lab Struct Biochem, Takustr 6, D-14195 Berlin, Germany.
ABSTRACT: lambda N-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein lambda N, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a lambda N-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and lambda N, validated by crosslinking/mass spectrometry.
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Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding
Scientific Reports. 2017. Scacioc, A et al. Max Planck Inst Biophys Chem, Res Grp Autophagy, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: PROPPINs (beta-propellers that bind polyphosphoinositides) are PtdIns3P and PtdIns(3,5)P-2 binding autophagy related proteins. They contain two phosphatidylinositolphosphate (PIP) binding sites and a conserved FRRG motif is essential for PIP binding. Here we present the 2.0 angstrom resolution crystal structure of the PROPPIN Atg18 from Pichia angusta. We designed cysteine mutants for labelling with the fluorescence dyes to probe the distances of the mutants to the membrane. These measurements support a model for PROPPIN-membrane binding, where the PROPPIN sits in a perpendicular or slightly tilted orientation on the membrane.
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Modeling protein excited-state structures from “over-length” chemical cross-links
Journal of Biological chemistry. 2017. Ding, YH et al. Natl Inst Biol Sci, 7 Sci Pk Rd,ZGC Life Sci Pk, Beijing 102206, Peoples R China.
ABSTRACT: Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones.
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SUMO-targeted DNA translocase Rrp2 protects the genome from Top2-induced DNA damage
Molecular Cell. 2017. Wei, Y et al. Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT: The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons.
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Structural basis for substrate selection by the translocation and assembly module of the β‐barrel assembly machinery
Molecular Microbiology. 2017. Rebecca S. Bamert, Karl Lundquist, Hyea Hwang, Chaille T. Webb et al. School of Physics, Georgia Institute of Technology, Atlanta, GA 30332, USA, Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
ABSTRACT: The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the -barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex.
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Conserved and unique features of the fission yeast core Atg1 complex
Autophagy. 2017. Nanji, T et al. 2350 Hlth Sci Mall, Vancouver, BC V6T 1Z3, Canada.
ABSTRACT: Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins.
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Characterizing protein dynamics with integrative use of bulk and single-molecule techniques
Biochemistry. 2017. Liu, Z et al. Chinese Acad Sci, Wuhan Inst Phys & Math, State Key Lab Magnet Resonance & Atom Mol Phys, CAS Key Lab Magnet Resonance Biol Syst, Wuhan 430071, Hubei, Peoples R China.
ABSTRACT: A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate.
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EB1-binding–myomegalin protein complex promotes centrosomal microtubules functions
PNAS. 2017. Bouguenina, H et al. Aix Marseille Univ, CNRS, Inst Paoli Calmettes, CRCM, F-13009 Marseille, France.
ABSTRACT: Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2.
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A new role for FBP21 as regulator of Brr2 helicase activity
Nucleic Acids Research. 2017. Henning, LM et al. Free Univ Berlin, Lab Prot Biochem, Inst Chem & Biochem, Thielallee 63, D-14195 Berlin, Germany.
ABSTRACT: Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2.
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Proteome-wide mapping of endogenous SUMOylation sites in mouse testis
Molecular & Cellular Proteomics. 2017. Cai, LL et al. Fudan Univ, Inst Biomed Sci, Sch Life Sci, Shanghai 200032, Peoples R China.
ABSTRACT: SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry.
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Combining chemical cross-linking and mass spectrometry of intact protein complexes to study the architecture of multi-subunit protein assemblies
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS. 2017. Haupt, C et al. Martin Luther Univ Halle Wittenberg, Interdisciplinary Res Ctr HALOmem, Halle, Germany.
ABSTRACT: Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
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Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide
Journal of Biological Chemistry. 2017. Blummel, AS et al. Univ Freiburg, Fac Med, ZBMZ, Inst Biochem & Mol Biol, D-79104 Freiburg, Germany.
ABSTRACT: Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event.
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Structural Insights of WHAMM's Interaction with Microtubules by Cryo-EM
Journal of Molecular Biology. 2017. Liu, TY et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China.
ABSTRACT: WASP homolog associated with actin, membranes, and microtubules (WHAMM) is a vertebrate protein functioning in membrane tubulation for intracellular membrane trafficking and specific organelle formation. Composed of multiple domains, WHAMM can bind to membrane and microtubule (MT) and promote actin polymerization nucleation. Previous work revealed that WHAMM's activity to promote actin nucleation is repressed upon binding to MTs. Here, we discovered that WHAMM interacts with alpha beta-tubulin through a small peptide motif within its MT-binding domain.
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Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins
PLOS ONE. 2017. Debelyy, MO et al. Univ Fribourg, Div Biochem, Dept Biol, Fribourg, Switzerland.
ABSTRACT: Here we made an attempt to obtain partial structural information on the topology of multi-span integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks.
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Protocol for analyzing protein ensemble structures from chemical cross-links using DynaXL
Biophysics Reports. 2017. Gong, Zhou et al. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, China
ABSTRACT: Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures.
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Structural basis for $\lambda$N-dependent processive transcription antitermination
Nature Microbiology. 2017. Said, N et al. Free Univ Berlin, Lab Struct Biochem, Takustr 6, D-14195 Berlin, Germany.
ABSTRACT: lambda N-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein lambda N, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a lambda N-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and lambda N, validated by crosslinking/mass spectrometry.
[more...]
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Structure of RNA polymerase bound to ribosomal 30S subunit
eLife. 2017. Demo, G et al. Univ Massachusetts, Sch Med, Dept Biochem & Mol Pharmacol, RNA Therapeut Inst, Worcester, MA 01655 USA.
ABSTRACT: In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit.
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An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly
EMBO JOURNAL. 2017. Jakhanwal, S et al. Max Planck Inst Biophys Chem, Dept Neurobiol, Gottingen, Germany.
ABSTRACT: Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18-1 as a possible acceptor complex for the R-SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18-1 remains tethered by the N-terminal domain of syntaxin1.
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Structural basis for substrate selection by the translocation and assembly module of the $\beta$-barrel assembly machinery
Molecular Microbiology. 2017. Rebecca S. Bamert, Karl Lundquist, Hyea Hwang, Chaille T. Webb et al. School of Physics, Georgia Institute of Technology, Atlanta, GA 30332, USA, Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia
ABSTRACT: The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the -barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex.
[more...]
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Acetylation and phosphorylation control both local and global stability of the chloroplast F 1 ATP synthase
Scientific Reports . 2017. Schmidt, C et al. Univ Oxford, Dept Chem, Oxford, England.
ABSTRACT: ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of epsilon and delta subunits.
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应用随机寡核苷酸文库修饰磁性颗粒提高血浆蛋白质的鉴定覆盖度
色谱. 2017. 邓 楠 et al. 中国科学院大连化学物理研究所, 中科院分离分析化学重点实验室, 国家色谱研究分析中心, 辽宁 大连 116023
ABSTRACT: A novel human plasma proteome sample pretreatment strategy was developed based on the interaction of random oligonucleotides with human plasma proteins, such as ionic inter. action, affinity interaction, hydrophobic interaction, hydrogen bonding or spatial structure and so on. Random oligonucleotide library was immobilized on the magnetic particles by biotin. avidin interaction, and dispersed in 20 mmol/L Tris. HCl buffer (pH 7.4), followed by incubation with plasma proteins. Two elution systems were used to elute the proteins interacted with random oligonucleotides, separately.
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Structural determinants of Neosartorya fischeri antifungal protein (NFAP) for folding, stability and antifungal activity
Scientific Reports . 2017. Galgoczy, L et al. Med Univ Innsbruck, Bioctr, Div Mol Biol, Innrain 80-82, A-6020 Innsbruck, Austria.
ABSTRACT: The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein.
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SpotLight Proteomics: uncovering the hidden blood proteome improves diagnostic power of proteomics
Scientific Reports. 2017. Lundstrom, SL et al. Karolinska Inst, Dept Med Biochem & Biophys, Div Physiol Chem 1, Stockholm, Sweden.
ABSTRACT: The human blood proteome is frequently assessed by protein abundance profiling using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). In traditional sequence database search, many good-quality MS/MS data remain unassigned. Here we uncover the hidden part of the blood proteome via novel SpotLight approach. This method combines de novo MS/MS sequencing of enriched antibodies and co-extracted proteins with subsequent label-free quantification of new and known peptides in both enriched and unfractionated samples.
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Combining de novo peptide sequencing algorithms, a synergistic approach to boost both identifications and confidence in bottom-up proteomics
Journal of Proteome Research. 2017. Blank-Landeshammer, B et al. ISAS eV, Leibniz Inst Analyt Wissensch, D-44139 Dortmund, Germany.
ABSTRACT: Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms.
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Comprehensive de novo peptide sequencing from MS/MS pairs generated through complementary collision induced dissociation and 351 nm ultraviolet photodissociation
Analytical chemistry. 2017. Horton, AP et al. Univ Texas Austin, Dept Mol Biosci, Ctr Syst & Synthet Biol, Austin, TX 78712 USA.
ABSTRACT: We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data.
[more...]
Use: pNovo



Comprehensive de Novo Peptide Sequencing from MS/MS Pairs Generated through Complementary Collision Induced Dissociation and 351 nm Ultraviolet …
Analytical chemistry. 2017. Horton, AP et al. Univ Texas Austin, Dept Mol Biosci, Ctr Syst & Synthet Biol, Austin, TX 78712 USA.
ABSTRACT: We describe a strategy for de novo peptide sequencing based on matched pairs of tandem mass spectra (MS/MS) obtained by collision induced dissociation (CID) and 351 nm ultraviolet photodissociation (UVPD). Each precursor ion is isolated twice with the mass spectrometer switching between CID and UVPD activation modes to obtain a complementary MS/MS pair. To interpret these paired spectra, we modified the UVnovo de novo sequencing software to automatically learn from and interpret fragmentation spectra, provided a representative set of training data.
[more...]
Use: pNovo



IgM antibodies against phosphorylcholine promote polarization of T regulatory cells from patients with atherosclerotic plaques; systemic lupus erythematosus and healthy donors
Atherosclerosis. 2017. Sun, JT et al. Karolinska Inst, Unit Immunol & Chron Dis, Inst Environm Med, Stockholm, Sweden.
ABSTRACT: Background and aims: IgM antibodies against phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE), where the risk of CVD and atherosclerosis is high. We here study the effects of IgM anti-PC immune regulation. Methods: Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, six SLE patients with age-and sex-matched controls, and symptom-giving human atherosclerotic plaques.
[more...]
Use: pNovo



Combining De Novo Peptide Sequencing Algorithms; A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics
Journal of Proteome Research. 2017. Blank-Landeshammer, B et al. ISAS eV, Leibniz Inst Analyt Wissensch, D-44139 Dortmund, Germany.
ABSTRACT: Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms.
[more...]
Use: pNovo



HILAQ: a novel strategy for newly synthesized protein quantification
Journal of proteome research. 2017. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT: Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol.
[more...]
Use: pQuant



HILAQ: A novel strategy for newly synthesized protein quantification
Journal of proteome research. 2017. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.
ABSTRACT: Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol.
[more...]
Use: pQuant



Immunopurification and mass spectrometry identifies protein phosphatase 2A (PP2A) and BIN2/GSK3 as regulators of AKS transcription factors in Arabidopsis
Molecular Plant. 2017. Bu, Shuo-Lei et al. Carnegie Inst Sci, Dept Plant Biol, 290 Panama St, Stanford, CA 94305 USA; Hebei Normal Univ, Hebei Collaborat Innovat Ctr Cell Signaling, Hebei Key Lab Mol & Cellular Biol, Coll Life Sci, Shijiazhuang 050024, Hebei, Peoples R China
ABSTRACT: ABA induces the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1,AKS2, and AKS3). The unphosphorylated AKSs facilitate stomatal opening through promoting the transcription of genes encoding inwardly rectifying K+ channels (Takahashi et al., 2013). AKS1 and AKS3 are also regulators of flowering (Ito et al., 2012). However, the kinases and phosphatases that directly control the phosphorylation status of AKSs in vivo have not been fully characterized.
[more...]
Use: pQuant



Association of IL-10-regulating microRNAs in peripheral blood mononuclear cells with the pathogenesis of autoimmune thyroid disease
Immunological Investigations. 2017. Takuse, Y et al. Osaka Univ, Grad Sch Med, Dept Biomed Informat, Div Hlth Sci, Yamadaoka 1-7, Suita, Osaka 5650871, Japan.
ABSTRACT: Interleukin (IL)-10 is known to suppress inflammation in autoimmune diseases. IL-10 can be regulated by miRNAs. To elucidate the involvement of miRNAs that regulate IL-10 expression with the pathogenesis of autoimmune thyroid disease (AITD), we examined the expression levels of hsa-miR-27a-3p, hsa-miR-98-5p, hsa-miR-106a-5p, and hsamiR- 223-3p in peripheral blood mononuclear cells (PBMCs) from 43 patients with Graves' disease (GD), 38 patients with Hashimoto's disease (HD), and 21 healthy volunteers.
[more...]
Use: pMatch



Informed-Proteomics: open-source software package for top-down proteomics
Nature Methods. 2017. Park, J et al. Pacific Northwest Natl Lab, Biol Sci Div, Richland, WA 99354 USA.
ABSTRACT: Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography-tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed.
[more...]
Use: pTop




2016




Quantitative profiling of the activity of protein lysine methyltransferase SMYD2 using SILAC-based proteomics
Molecular & Cellular Poteomics. 2016. Olsen, JB et al. Lilly USA, Lilly Res Labs, Indianapolis, IN 46285 USA.
ABSTRACT: The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell.
[more...]
Use: pFind



Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics
Electrophoresis. 2016. Li, SS et al. Nankai Univ, Coll Pharm, State Key Lab Med Chem Biol, Tianjin, Peoples R China.
ABSTRACT: O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac(4)GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells.
[more...]
Use: pFind; pBuild



Global post-translational modification discovery
Journal of Proteome Research. 2016. Qiyao Li et al. Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI, 53706;
ABSTRACT: A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples.
[more...]
Use: pFind



A novel quantitative mass spectrometry platform for determining protein O-GlcNAcylation dynamics
Molecular & Cellular Proteomics. 2016. Wang, XS et al. Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Program, Room 9-124,3400 Civ Ctr Blvd,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT: Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing C-13-labeled UDP-GlcNAc from C-13(6)-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates.
[more...]
Use: pXtract; pParse; pFind



Deep coverage proteomics identifies more low-abundance missing proteins in human testis tissue with Q-exactive HF mass spectrometer
Journal of Proteome Research. 2016. Wei, W et al. Beijing Inst Radiat Med, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, Natl Engn Res Ctr Prot Drugs,State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described.
[more...]
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ABRF Proteome Informatics Research Group (iPRG) 2015 Study: detection of differentially abundant proteins in label-free quantitative LC–MS/MS experiments
Journal of Proteome Research. 2016. Choi, M et al. Northeastern Univ, Boston, MA 02115 USA.
ABSTRACT: Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results.
[more...]
Use: pFind; pQuant



GALNT14 genotype effectively predicts the therapeutic response in unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization
Pharmacogenomics. 2016. Liang, KH et al. Chang Gung Mem Hosp, Liver Res Ctr, 5 Fu Sing St, Taoyuan, Taiwan.
ABSTRACT: Aim: Transcatheter arterial chemoembolization is currently the standard treatment in hepatocellular carcinoma patients with Barcelona Clinic Liver Cancer stage B. Genomic variants of GALNT14 were recently identified as effective predictors for chemotherapy responses in Barcelona Clinic Liver Cancer stage C patients. Methods: We investigated the prognosis predictive value of GALNT14 genotypes in 327 hepatocelluar carcinoma patients treated by transcatheter arterial chemoembolization. Result: Cox proportional hazards model analysis showed that the genotype 'TT' was associated with shorter time-to-response (multivariate p < 0.001), time-to-complete-response (p = 0.004) and longer time-to-tumor progression (p < 0.001), compared with the genotype 'non-TT'.
[more...]
Use: pFind



Searching missing proteins based on the optimization of membrane protein enrichment and digestion process
Journal of Proteome Research. 2016. Zhao, MZ et al. Beijing Inst Radiat Med, Natl Engn Res Ctr Prot Drugs, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing,State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: A membrane protein enrichment method composed of ultracentrifugation and detergent-based extraction was first developed based on MCF7 cell line. Then, in-solution digestion with detergents and eFASP (enhanced filter-aided sample preparation) with detergents were compared with the time-consuming in-gel digestion method. Among the in-solution digestion strategies, the eFASP combined with RapiGest identified 1125 membrane proteins. Similarly, the eFASP combined with sodium deoxycholate identified 1069 membrane proteins; however, the in-gel digestion characterized 1091 membrane proteins.
[more...]
Use: pFind; pBuild



Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation
Journal of Proteomics. 2016. Ma, C et al. Georgia State Univ, Dept Chem, Ctr Diagnost & Therapeut, Atlanta, GA 30303 USA.
ABSTRACT: Core-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma.
[more...]
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Monitoring cellular phosphorylation signaling pathways into chromatin and down to the gene level
Molecular & Cellular Proteomics. 2016. Han, YM et al. Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Program,Smilow Ctr Translat Res, 3400 Civ Ctr Blvd,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT: Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways.
[more...]
Use: pQuant; pFind



An NGS-independent strategy for proteome-wide identification of single amino acid polymorphisms by mass spectrometry
Analytical Chemistry. 2016. Xiong, Y et al. Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Key Lab Syst Microbial Biotechnol, Tianjin 300308, Peoples R China.
ABSTRACT: Detection of proteins containing single amino acid polymorphisms (SAPs) encoded by nonsynonymous SNPs (nsSNPs) can aid researchers in studying the functional significance of protein variants. Most proteogenomic approaches for large-scale SAPs mapping require construction of a sample specific database containing protein variants predicted from the next-generation sequencing (NGS) data. Searching shotgun proteomic data sets against these NGS-derived databases allowed for identification of SAP peptides, thus validating the proteome-level sequence variation.
[more...]
Use: pFind



Application of in-house virtual protein database performed in genomic-proteomic combined research on heavy-metal stressed onion roots
Biotechnology letters. 2016. Ning, Chanjuan et al. South China Normal Univ, Sch Life Sci, Key Lab Ecol & Environm Sci Guangdong Higher Educ, Guangzhou 510631, Guangdong, Peoples R China
ABSTRACT: To establish an in-house virtual protein database that can be employed in proteomic research on non-model plants.A total of 87,430 unigenes were obtained through transcriptome sequencing from onion roots. Of these, 24,305 unigenes were annotated and their nucleotide sequences of coding regions were translated into amino acid sequences. The corresponding 24,305 amino acid sequences were considered as an in-house virtual protein database. Thirty-two protein spots with significant differential abundance were selected.
[more...]
Use: pFind



The Arabidopsis B-box protein BZS1/BBX20 interacts with HY5 and mediates strigolactone regulation of photomorphogenesis
Journal of Genetics and Genomics. 2016. Wei, CQ et al. Hebei Normal Univ, Hebei Key Lab Mol & Cellular Biol, Key Lab Mol & Cellular Biol,Coll Life Sci, Hebei Collaborat Innovat Ctr Cell Signaling,Minis, Shijiazhuang 050024, Peoples R China.
ABSTRACT: Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR)-signaling pathway and a positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins, including light-signaling components COP1 and HY5.
[more...]
Use: pQuant; pFind



ABRF Proteome Informatics Research Group (iPRG) 2015 Study: Detection of Differentially Abundant Proteins in Label-Free Quantitative LC--MS/MS Experiments
Journal of Proteome Research. 2016. Choi, M et al. Northeastern Univ, Boston, MA 02115 USA.
ABSTRACT: Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results.
[more...]
Use: pFind; pQuant



Global proteomics analysis of protein lysine methylation
Current Protocols in Protein Science. 2016. Xing-Jun Cao1; Benjamin A. Garcia et al. Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT: Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites.
[more...]
Use: pFind



Immuno-purification mass spectrometry identifies protein phosphatase 2A (PP2A) and BIN2/GSK3 as regulators of AKS transcription factors in Arabidopsis
Molecular Plant. 2016. Shuo-Lei Bu; Chao Liu; Ning Liu et al. Hebei Key Laboratory of Molecular and Cellular Biology, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Hebei 050024, China | Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA
ABSTRACT:
Use: pFind; pQuant



Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides
Analytical and Bioanalytical Chemistry. 2016. Zhu, H et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.
ABSTRACT: N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment.
[more...]
Use: pFind; pBuild



Triptolide induces cell killing in multidrug-resistant tumor cells via CDK7/RPB1 rather than XPB or p44
Molecular Cancer Therapeutics. 2016. Yi, JM et al. Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Div Antitumor Pharmacol, Shanghai, Peoples R China.
ABSTRACT: Multidrug resistance (MDR) is a major cause of tumor treatment failure; therefore, drugs that can avoid this outcome are urgently needed. We studied triptolide, which directly kills MDR tumor cells with a high potency and a broad spectrum of cell death. Triptolide did not inhibit P-glycoprotein (P-gp) drug efflux and reduced P-gp and MDR1 mRNA resulting from transcription inhibition. Transcription factors including c-MYC, SOX-2, OCT-4, and NANOG were not correlated with triptolide-induced cell killing, but RPB1, the largest subunit of RNA polymerase II, was critical in mediating triptolide's inhibition of MDR cells.
[more...]
Use: pFind



A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses
Journal of Proteomics. 2016. Xiong, YH et al. Chinese Acad Agr Sci, State Key Lab Biol Plant Dis & Insect Pests, Inst Plant Protect, Beijing 100193, Peoples R China.
ABSTRACT: Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in the gene transcription regulation. Here, we report high quality proteome-scale data for lysine-acetylation (Mac) sites and Mac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Mac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chioroplast. Seven putative acetylation motifs were detected.
[more...]
Use: pFind



A receptor heteromer mediates the male perception of female attractants in plants
Nature. 2016. Wang, T et al. Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol & Dev Biol, Beijing 100101, Peoples R China.
ABSTRACT: Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth. Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube(1-3), the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1-MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana.
[more...]
Use: pLink



Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain
Journal of Proteomics. 2016. Hung, CW et al. Univ Kiel, Inst Expt Med, Systemat Prote & Bioanalyt, Kiel, Germany.
ABSTRACT: Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications.
[more...]
Use: pLink



Molecular architecture of SF3b and structural consequences of its cancer-related mutations
Molecular Cell. 2016. Cretu, C et al. Max Planck Inst Biophys Chem, Res Grp Macromol Crystallog, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform.
[more...]
Use: pLink



The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2016. Kloet, SL et al. Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Fac Sci, Dept Mol Biol, Nijmegen, Netherlands.
ABSTRACT: Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites.
[more...]
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Structure and function of the nuclear pore complex cytoplasmic mRNA export platform
Cell. 2016. Fernandez-Martinez, J et al. Rockefeller Univ, Lab Cellular & Struct Biol, New York, NY 10065 USA.
ABSTRACT: The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed.
[more...]
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Molecular architecture of the Saccharomyces cerevisiae activated spliceosome
Science. 2016. Reinhard Rauhut, Patrizia Fabrizio, Olexandr Dybkov et al. Department of Cellular Biochemistry, Max Planck Institute (MPI) for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
ABSTRACT: The activated spliceosome (B-act) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae B-act complex at 5.8-angstrom resolution. Our model reveals that in B-act, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. 
[more...]
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Molecular architecture of the human U4/U6. U5 tri-snRNP
Science. 2016. Dmitry E. Agafonov, Berthold Kastner, Olexandr Dybkov et al. Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
ABSTRACT: The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position.
[more...]
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Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes
NATURE. 2016. Wu, S et al. Tsinghua Univ, Sch Life Sci, Beijing Adv Innovat Ctr Struct Biol, Minist Educ,Key Lab Prot Sci, Beijing 100084, Peoples R China.
ABSTRACT: Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm(1,2). Hundreds of assembly factors, organized into sequential functional groups(3,4), facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown.
[more...]
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The 3.8 Å structure of the U4/U6. U5 tri-snRNP: Insights into spliceosome assembly and catalysis
SCIENCE. 2016. Wan, RX et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Minist Educ,Key Lab Prot Sci, Beijing 100084, Peoples R China.
ABSTRACT: Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy.
[more...]
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Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin
Nature communications. 2016. Hiragami-Hamada, K et al. Max Planck Inst Biophys Chem, Lab Chromatin Biochem, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT: Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1 beta is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces.
[more...]
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Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
eLife. 2016. Tan, D et al. Chinese Acad Med Sci, Peking Union Med Coll, Grad Program, Beijing 100730, Peoples R China.
ABSTRACT: To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy.
[more...]
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Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation
Nature chemical biology. 2016. Pao, KC et al. Univ Dundee, Med Res Council Prot Phosphorylat & Ubiquitylat U, Dundee, Scotland.
ABSTRACT: E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation.
[more...]
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UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly
Nature Communications. 2016. Hunziker, M et al. Rockefeller Univ, Lab Prot & Nucle Acid Chem, New York, NY 10065 USA.
ABSTRACT: Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 50 end of pre-ribosomal RNA.
[more...]
Use: pLink



Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System
PLOS Pathogens. 2016. Gorasia, DG et al. Univ Melbourne, Melbourne Dent Sch, Oral Hlth CRC, Inst Bio21, Melbourne, Vic, Australia.
ABSTRACT: The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown.
[more...]
Use: pLink



Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length
NATURE COMMUNICATIONS. 2016. Zheng, XD et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Med, Dept Basic Med Sci, Beijing 100084, Peoples R China.
ABSTRACT: Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets beta-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's alpha-beta surface of beta-tubulin.
[more...]
Use: pLink



Structural basis for receptor recognition and pore formation of a zebrafish aerolysin‐like protein
EMBO REPORTS. 2016. Jia, N et al. Univ Sci & Technol China, Hefei Natl Lab Phys Sci Microscale, Hefei 230026, Peoples R China.
ABSTRACT: Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a -prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore.
[more...]
Use: pLink



Cross-linking immunoprecipitation-MS (xIP-MS): topological analysis of chromatin-associated protein complexes using single affinity purification
Molecular & Cellular Proteomics. 2016. Makowski, MM et al. Radboud Inst Mol Life Sci, Geert Grootepl 28, NL-6525 GA Nijmegen, Netherlands.
ABSTRACT: In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis.
[more...]
Use: pLink



Rational design of a protein that binds integrin αvβ3 outside the ligand binding site
Nature Communications. 2016. Turaga, Ravi Chakra et al. Georgia State Univ, Dept Biol, POB 4010, Atlanta, GA 30303 USA
ABSTRACT: Integrin alpha(v)beta(3) expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin alpha(v)beta(3) outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin alpha(v)beta(3)-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin alpha(v)beta(3). ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature.
[more...]
Use: pLink



Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement
EMBO reports. 2016. Setiaputra, DT et al. Univ British Columbia, Dept Biochem & Mol Biol, Vancouver, BC, Canada.
ABSTRACT: Elongator is a 850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold.
[more...]
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The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction
Elife. 2016. Fourmann, JB et al. Max Planck Inst Biophys Chem, Dept Cellular Biochem, Gottingen, Germany.
ABSTRACT: The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1's G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1's C-terminal-domain (CTD) and Ntr2.
[more...]
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CryoEM structure of yeast cytoplasmic exosome complex
Cell research. 2016. Liu, JJ et al. Tsinghua Univ, Minist Educ Key Lab Prot Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing Adv Innovat Ctr Struct Biol,Sch Life Sci, Beijing 100084, Peoples R China.
ABSTRACT: The eukaryotic multi-subunit RNA exosome complex plays crucial roles in 3'-to-5' RNA processing and decay. Rrp6 and Ski7 are the major cofactors for the nuclear and cytoplasmic exosomes, respectively. In the cytoplasm, Ski7 helps the exosome to target mRNAs for degradation and turnover via a through-core pathway. However, the interaction between Ski7 and the exosome complex has remained unclear. The transaction of RNA substrates within the exosome is also elusive. In this work, we used single-particle cryo-electron microscopy to solve the structures of the Ski7-exosome complex in RNA-free and RNA-bound forms at resolutions of 4.2 angstrom and 5.8 angstrom, respectively.
[more...]
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Increasing the depth of mass-spectrometry-based structural analysis of protein complexes through the use of multiple cross-linkers
Analytical Chemistry. 2016. Ding, YH et al. Nat Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT: Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS3 and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins.
[more...]
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The MTA1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4/7
Protein Science. 2016. Schmidberger, JW et al. Univ Sydney, Sch Life & Environm Sci, Sydney, NSW 2006, Australia.
ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4.
[more...]
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Enrichment of cross-linked peptides using charge-based fractional diagonal chromatography (ChaFRADIC)
Journal of proteome research. 2016. Tinnefeld, V et al. Leibniz Inst Analyt Wissensch ISAS eV, D-44227 Dortmund, Germany.
ABSTRACT: Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment.
[more...]
Use: pLink



Xilmass: a new approach toward the identification of cross-linked peptides
Analytical Chemistry. 2016. Yilmaz, S et al. VIB, Med Biotechnol Ctr, B-9000 Ghent, Belgium.
ABSTRACT: Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, crosS-linking complements several structural determination approaches such as cryo:EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair.
[more...]
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The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core
Nature Communications. 2016. Rajasekar, KV et al. Univ Oxford, Dept Biochem, South Parks Rd, Oxford OX1 3QU, England.
ABSTRACT: Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor sR preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex.
[more...]
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The Architecture of Trypanosoma brucei editosomes
PNAS. 2016. McDermott, SM et al. Ctr Infect Dis Res, Seattle, WA 98109 USA.
ABSTRACT: Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. Editing is catalyzed by three distinct similar to 20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified.
[more...]
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ECL: an exhaustive search tool for the identification of cross-linked peptides using whole database
BMC bioinformatics. 2016. Yu, FC et al. Hong Kong Univ Sci & Technol, Div Biomed Engn, Hong Kong, Hong Kong, Peoples R China.
ABSTRACT: Background: Chemical cross-linking combined with mass spectrometry (CX-MS) is a high-throughput approach to studying protein-protein interactions. The number of peptide-peptide combinations grows quadratically with respect to the number of proteins, resulting in a high computational complexity. Widely used methods including xQuest (Rinner et al., Nat Methods 5(4): 315-8, 2008; Walzthoeni et al., Nat Methods 9(9): 901-3, 2012), pLink (Yang et al., Nat Methods 9(9): 904-6, 2012), ProteinProspector (Chu et al., Mol Cell Proteomics 9: 25-31, 2010; Trnka et al., 13(2): 420-34, 2014) and Kojak (Hoopmann et al., J Proteome Res 14(5): 2190-198, 2015) avoid searching all peptide-peptide combinations by pre-selecting peptides with heuristic approaches.
[more...]
Use: pLink



Pex17p-dependent assembly of Pex14p/Dyn2p-subcomplexes of the peroxisomal protein import machinery
European Journal of Cell Biology. 2016. Chan, A et al. Ruhr Univ Bochum, Inst Biochem & Pathobiochem, Univ Str 150, D-44780 Bochum, Germany.
ABSTRACT: Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive. In order to characterize the function of Pex17p, we compared the composition and size of peroxisomal receptor-docking complexes from wild-type and pex17 Delta cells.
[more...]
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An open data format for visualization and analysis of cross-linked mass spectrometry results
Journal of the American Society for Mass Spectrometry. 2016. Hoopmann, MR et al. Inst Syst Biol, Seattle, WA 98109 USA.
ABSTRACT: Protein-protein interactions are an important element in the understanding of protein function, and chemical cross-linking shotgun mass spectrometry is rapidly becoming a routine approach to identify these specific interfaces and topographical interactions. Protein cross-link data analysis is aided by dozens of algorithm choices, but hindered by a lack of a common format for representing results. Consequently, interoperability between algorithms and pipelines utilizing chemical cross-linking remains a challenge.
[more...]
Use: pLink



Structural characterization of coatomer in its cytosolic state
Protein & Cell. 2016. Wang, SL et al. Chinese Acad Sci, Inst Biophys, Natl Key Lab Biomacromol, Beijing 100101, Peoples R China.
ABSTRACT: Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting.
[more...]
Use: pLink



Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation
PROTEIN & CELL. 2016. Ma, CY et al. Tsinghua Univ, Sch Life Sci, Beijing 100084, Peoples R China.
ABSTRACT: The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast.
[more...]
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ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry
Analytical and Bioanalytical Chemistry. 2016. Wen, YR et al. Univ Ghent, Unit Biol Mass Spectrometry & Prote, Lab Prot Biochem & Biomol Engn L ProBE, KL Ledeganckstr 35, B-9000 Ghent, Belgium.
ABSTRACT: Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response.
[more...]
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Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis
RNA. 2016. Remus, BS et al. Sloan Kettering Inst, Program Mol Biol, New York, NY 10065 USA.
ABSTRACT: Yeast tRNA ligase (TrI1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. TrI1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. TrI1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the TrI1 ligase domain in mammalian proteomes.
[more...]
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Rational design of a protein that binds integrin $\alpha$v$\beta$3 outside the ligand binding site
Nature Communications. 2016. Turaga, Ravi Chakra et al. Georgia State Univ, Dept Biol, POB 4010, Atlanta, GA 30303 USA
ABSTRACT: Integrin alpha(v)beta(3) expression is altered in various diseases and has been proposed as a drug target. Here we use a rational design approach to develop a therapeutic protein, which we call ProAgio, that binds to integrin alpha(v)beta(3) outside the classical ligand-binding site. We show ProAgio induces apoptosis of integrin alpha(v)beta(3)-expressing cells by recruiting and activating caspase 8 to the cytoplasmic domain of integrin alpha(v)beta(3). ProAgio also has anti-angiogenic activity and strongly inhibits growth of tumour xenografts, but does not affect the established vasculature.
[more...]
Use: pLink



Application of de novo sequencing to large-scale complex proteomics data sets
Journal of Proteome Research. 2016. Arun Devabhaktuni et al. Department of Chemical & Systems Biology, United States
ABSTRACT: Dependent on concise, predefined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results. Here, we present novel metrics for benchmarking de novo sequencing algorithm performance on large-scale proteomics data sets and present a method for accurately calibrating false discovery rates on de novo results.
[more...]
Use: pNovo



Peptide de novo sequencing of mixture tandem mass spectra
Proteomics. 2016. Vladimir Gorshkov et al. University of Southern Denmark Odense M
ABSTRACT: The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications.
[more...]
Use: pNovo



De novo peptide sequencing using CID and HCD spectra pairs
Proteomics. 2016. Yan, Y et al. Univ Saskatchewan, Dept Mech Engn, 57 Campus, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible, including, collision-induced dissociation (CID) higher energy collisional dissociation (HCD), electron-capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs.
[more...]
Use: pNovo



Human IgM Antibodies to Malondialdehyde Conjugated With Albumin Are Negatively Associated With Cardiovascular Disease Among 60-Year-Olds
Journal of the American Heart Association. 2016. Thiagarajan, D et al. Karolinska Inst, Inst Environm Med, Nobels Vag 13, S-17165 Stockholm, Sweden.
ABSTRACT: Background-Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low-density lipoprotein, but antibodies against oxidized low-density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti-MDA). Methods and Results-In a 5- to 7-year follow-up of 60-year-old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age-and sex-matched controls were tested for IgM anti-MDA by ELISA.
[more...]
Use: pNovo



Application of de Novo Sequencing to Large-Scale Complex Proteomics Data Sets
Journal of Proteome Research. 2016. Arun Devabhaktuni et al. Department of Chemical & Systems Biology, United States
ABSTRACT: Dependent on concise, predefined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results. Here, we present novel metrics for benchmarking de novo sequencing algorithm performance on large-scale proteomics data sets and present a method for accurately calibrating false discovery rates on de novo results.
[more...]
Use: pNovo



Structure of the voltage-gated calcium channel Cav1. 1 at 3.6 {\AA} resolution
Nature. 2016. Wu, Jianping et al. Tsinghua Univ, Sch Life Sci, State Key Lab Membrane Biol, Beijing 100084, Peoples R China|Tsinghua Univ, Sch Med, Beijing 100084, Peoples R China|Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, Beijing 100084, Peoples R China|Tsinghua Univ, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing 100084, Peoples R China;
ABSTRACT: The coverage and throughput of data-independent acquisition (DIA)-based phosphoproteomics is limited by its dependence on experimental spectral libraries. Here the authors develop a DIA workflow based on in silico spectral libraries generated by a novel deep neural network to expand phosphoproteome coverage.Phosphoproteomics integrating data-independent acquisition (DIA) enables deep phosphoproteome profiling with improved quantification reproducibility and accuracy compared to data-dependent acquisition (DDA)-based phosphoproteomics.
[more...]
Use: pLink; pParse




2015




Controlling nonspecific trypsin cleavages in LC-MS/MS-based shotgun proteomics using optimized experimental conditions
Analyst. 2015. Fang, P et al. Fudan Univ, Minhang Hosp, Shanghai 201199, Peoples R China.
ABSTRACT: Trypsin has traditionally been used for enzymatic digestion during sample preparation in shotgun proteomics. The stringent specificity of trypsin is essential for accurate protein identification and quantification. But nonspecific trypsin cleavages are often observed in LC-MS/MS-based shotgun proteomics. To explore the extent of nonspecific trypsin cleavages, a series of biological systems including a standard protein mixture, Saccharomyces cerevisiae, human serum, human cancer cell lines and mouse brain were examined.
[more...]
Use: pFind



A paired ions scoring algorithm based on Morpheus for simultaneous identification and quantification of proteome samples prepared by isobaric peptide termini labeling strategies
Proteomics. 2015. Zhang, Shen et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, Key Lab,Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: The isobaric peptide termini labeling (IPTL) method is a promising strategy in quantitative proteomics for its high accuracy, while the increased complexity of MS2 spectra originated from the paired b, y ions has adverse effect on the identification and the coverage of quantification. Here, a paired ions scoring algorithm (PISA) based on Morpheus, a database searching algorithm specifically designed for high-resolution MS2 spectra, was proposed to address this issue. PISA was first tested on two 1:1 mixed IPTL datasets, and increases in peptide to spectrum matchings, distinct peptides and protein groups compared to Morpheus itself and MASCOT were shown.
[more...]
Use: pFind



Integrated SDS removal and protein digestion by hollow fiber membrane based device for SDS-assisted proteome analysis
Talanta. 2015. Xia, SM et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China.
ABSTRACT: In this work, a novel integrated sample preparation device for SDS-assisted proteome analysis was developed, by which proteins dissolved in 4% (w/v) SDS were first diluted by 50% methanol, and then SDS was online removed by a hollow fiber membrane interface (HFMI) with 50 mM ammonium bicarbonate (pH 8.0) as an exchange buffer, finally digested by an immobilized enzyme reactor (IMER). To evaluate the performance of such an integrated device, bovine serum albumin dissolved in 4% (w/v) SDS as a model sample was analyzed; it could be found that similar to that obtained by direct analysis of BSA digests without SDS (the sequence coverage of 60.3 +/- 1.0%, n=3), with HFMI as an interface for SDS removal, BSA was identified with the sequence coverage of 61.0 +/- 1.0% (n=3).
[more...]
Use: pXtract; pBuild; pFind



Particulate capillary precolumns with double-end polymer monolithic frits for on-line peptide trapping and preconcentration
Chinese Chemical Letters. 2015. Xia, SM et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China.
ABSTRACT: In this work, a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed. Firstly, the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary (100 mu m i.d.). Subsequently, C18 particles (5 mu m, 100 angstrom) were packed into the capillary, and sealed with the polymer monolithic frit at another end.
[more...]
Use: pXtract; pBuild; pFind



Towards elucidating the stability, dynamics and architecture of the nucleosome remodeling and deacetylase complex by using quantitative interaction proteomics
The Federation of European Biochemical Societies Journal. 2015. Kloet, SL et al. Dept Mol Biol, M850-3-79 259 RIMLS,POB 9101, NL-6500 HB Nijmegen, Netherlands.
ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex is an evolutionarily conserved chromatin-associated protein complex. Although the subunit composition of the mammalian complex is fairly well characterized, less is known about the stability and dynamics of these interactions. Furthermore, detailed information regarding protein-protein interaction surfaces within the complex is still largely lacking. Here, we show that the NuRD complex interacts with a number of substoichiometric zinc finger-containing proteins.
[more...]
Use: pLink; pFind



The ratcheted and ratchetable structural states of RNA polymerase underlie multiple transcriptional functions
Molecular Cell. 2015. Sekine, S et al. RIKEN Syst & Struct Biol Ctr, Tsurumi Ku, 1-7-22 Suehiro Cho, Yokohama, Kanagawa 2300045, Japan.
ABSTRACT: DNA-dependent RNA polymerase (RNAP) accomplishes multiple tasks during transcription by assuming different structural forms. Reportedly, the "tight" form performs nucleotide addition to nascent RNA, while the "ratcheted" form is adopted for transcription inhibition. In this study, we performed Cys-pair crosslinking (CPX) analyses of various transcription complexes of a bacterial RNAP and crystallographic analyses of its backtracked and Gre-factor-bound states to clarify which of the two forms is adopted.
[more...]
Use: pLink; pFind



Large-scale identification of core-fucosylated glycopeptide sites in pancreatic cancer serum using mass spectrometry
Journal of Proteome Research. 2015. Tan, ZJ et al. Univ Michigan, Dept Surg, Ann Arbor, MI 48109 USA.
ABSTRACT: Glycosylation has significant effects on protein function and cell metastasis, which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to that of nonglycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting nonglycopeptides due to low ionization efficiency. Selective enrichment of glycopeptides from complex serum samples is essential for mass spectrometry (MS)-based analysis.
[more...]
Use: pFind



Unique diversity of the venom peptides from the scorpion Androctonus bicolor revealed by transcriptomic and proteomic analysis
Journal of proteomics. 2015. Zhang, L et al. China Univ Geosci, Dept Biol Sci & Technol, Sch Environm Studies, Wuhan 430074, Peoples R China.
ABSTRACT: Androctonus bicolor is one of the most poisonous scorpion species in the world. However, little has been known about the venom composition of the scorpion. To better understand the molecular diversity and medical significance of the venom from the scorpion, we systematically analyzed the venom components by combining transcriptomic and proteomic surveys. Random sequencing of 1000 clones from a cDNA library prepared from the venom glands of the scorpion revealed that 70% of the total transcripts code for venom peptide precursors.
[more...]
Use: pFind



Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom‐up proteomics PTM analysis
Proteomics. 2015. Sidoli, S et al. Univ Penn, Dept Biochem & Biophys, Perelman Sch Med, Philadelphia, PA 19104 USA.
ABSTRACT: MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events.
[more...]
Use: pFind; pBuild



Tissue-based proteogenomics reveals that human testis endows plentiful missing proteins
Journal of Proteome Research. 2015. Zhang, Yao et al. BGI Shenzhen, Shenzhen 518083, Peoples R China
ABSTRACT: Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals. Proteins were extracted and separated with glycine- and tricine-SDS-PAGE. A total of 9597 protein groups were identified; of these, 166 protein groups were listed as MPs, including 138 groups (83.1%) with transcriptional evidence.
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Identification of missing proteins defined by chromosome-centric proteome project in the cytoplasmic detergent-insoluble proteins
Journal of Proteome Research. 2015. Chen, Yang et al. Jinan Univ, Coll Life Sci & Technol, Inst Life & Hlth Engn, Key Lab Funct Prot Res Guangdong Higher Educ Inst, Guangzhou 510632, Guangdong, Peoples R China
ABSTRACT: Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2-4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins.
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Analysis of histones H3 and H4 reveals novel and conserved post-translational modifications in sugarcane
PLOS ONE. 2015. Moraes, I et al. Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-01498 Sao Paulo, Brazil.
ABSTRACT: Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate.
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Identification of HPV integration and gene mutation in HeLa cell line by integrated analysis of RNA-Seq and MS/MS data
Journal of proteome research. 2015. Sun, Han et al. Shanghai Acad Sci & Technol, Shanghai Ctr Bioinformat Technol, 1278 Ke Yuan Rd, Shanghai 201203, Peoples R China
ABSTRACT: HeLa cell line, which was derived from cervical carcinoma, provides an idea platform to study both the integration of human papillomavirus and the massive mutations occurring on the cancer cell genome. Proteogenomics is a field with the intersection of proteomics and genomics to perform gene annotation and identify gene mutation. In this work, we first identified the SNV/INDEL, structural variation (SV), and virus infection/integration events from RNA-Seq data of HeLa cell line; then, by applying proteogenomics strategy, we were able to detect some of the genomic events with the tandem mass spectrometry (MS/MS) data from the same sample.
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Finding missing proteins from the epigenetically manipulated human cell with stringent quality criteria
Journal of Proteome Research. 2015. Yang, Lijuan et al. Fudan Univ, Inst Biomed Sci, Dept Cell Biol, Key Lab Epigenet, Shanghai 200032, Peoples R China
ABSTRACT: The chromosome-centric human proteome project (C-HPP) has made great progress of finding protein evidence (PE) for missing proteins (PE2-4 proteins defined by the neXtProt), which now becomes an increasingly challenging field. As a majority of samples tested in this field were from adult tissues/cells, the developmental stage specific or relevant proteins could be missed due to biological source availability. We posit that epigenetic interventions may help to partially bypass such a limitation by stimulating the expression of the "silenced" genes in adult cells, leading to the increased chance of finding missing proteins.
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Special enrichment strategies greatly increase the efficiency of missing proteins identification from regular proteome samples
Journal of Proteome Research. 2015. Su, N et al. Beijing Proteome Res Ctr, 33 Sci Pk Rd, Beijing 102206, Peoples R China.
ABSTRACT: As part of the Chromosome-Centric Human Proteome Project (C-HPP) mission, laboratories all over the world have tried to map the entire missing proteins (MPs) since 2012. On the basis of the first and second Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we developed systematic enrichment strategies to identify MPs that fell into four classes: (1) low molecular weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), and (4) nucleic acid-associated proteins.
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Proteomic analysis of Allium cepa var. agrogarum L. roots under copper stress
PLANT AND SOIL. 2015. Qin, R et al. S China Normal Univ, Sch Life Sci, Key Lab Ecol & Environm Sci Guangdong Higher Educ, Guangzhou 510631, Guangdong, Peoples R China.
ABSTRACT: In the present study, the effects of Cu (2.0 and 8.0 mu M) on root growth of Allium cepa var. agrogarum L. were addressed and protein abundance levels were analyzed using the technology of proteomics combined with transcriptomics, in order to go deeper into the understanding of the mechanism of Cu toxicity on plant root systems at the protein level and to provide valuable information for monitoring and forecasting the effects of exposure to Cu in real scenarios conditions. Protein extraction; Two-dimensional electrophoresis (2-DE) analysis; Mass spectrometry analysis; Establishment of the in-house database; Restriction enzyme map of the in-house database and protein identification.
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freeQuant: a mass spectrometry label-free quantification software tool for complex proteome analysis
TheScientificWorldJournal. 2015. Deng, Ning et al. Department of Biomedical Engineering, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, China
ABSTRACT: Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity.
[more...]
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Experimental validation of Bacillus anthracis A16R proteogenomics
Scientific Reports. 2015. Gao, ZQ et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, Natl Engn Res Ctr Prot Drugs, State Key Lab Prote,Natl Ctr Prot Sci, 27 Taiping Rd, Beijing 100850, Peoples R China.
ABSTRACT: Anthrax, caused by the pathogenic bacterium Bacillus anthracis, is a zoonosis that causes serious disease and is of significant concern as a biological warfare agent. Validating annotated genes and reannotating misannotated genes are important to understand its biology and mechanisms of pathogenicity. Proteomics studies are, to date, the best method for verifying and improving current annotations. To this end, the proteome of B. anthracis A16R was analyzed via one-dimensional gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
[more...]
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Appraisal of the missing proteins based on the mRNAs bound to ribosomes
Journal of Proteome Research. 2015. Xu, Shaohang et al. BGI Shenzhen, Beishan Ind Zone, 11 Build, Shenzhen 518083, Peoples R China
ABSTRACT: Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins.
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Evaluation and Comparison of Aligners for De Novo Sequencing
International Journal of Advanced Computer Technology. 2015. Simin Zhu et al. Yunnan Minzu University, Kunming
ABSTRACT: In high-throughput proteomics research of tandem mass spectrometry, de novo sequencing provides a novel method to interpret MS/MS data without any help of sequence database and discover new organisms. In this paper, we have systematically evaluated and compared the capability of mainstream de novo sequencing software via testing data sets which have been correctly identified by Mascot and Sequest, so we can intuitively find out the optimal de novo sequencing software for protein identification.
Use: pNovo; pFind



一种结合生物医学知识的蛋白质组非标记定量分析方法及其应用
PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS. 2015. 潘超 et al. 浙江大学生物医学工程与仪器科学学院,生物医学工程教育部重点实验室
ABSTRACT: Label-free quantitative approach based mass spectrometry was used for analysis of complex proteomes, meanwhile, a method based on quantitative analysis which is used for explaining functions and interactions in a large-scale manner is of great importance. To systematically overcome this challenge, we should build a method combing with quantitation and qualification. We used Normalized Spectral Abundance Factor (NSAF) based peptide count as starting point for our analysis and proposed a new method with shared peptides to accurately evaluate abundance of Isoforms for complex proteomes.
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CyanOmics: an integrated database of omics for the model cyanobacterium Synechococcus sp. PCC 7002
DATABASE-THE JOURNAL OF BIOLOGICAL DATABASES AND CURATION. 2015. Yang, YH et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT: Cyanobacteria are an important group of organisms that carry out oxygenic photosynthesis and play vital roles in both the carbon and nitrogen cycles of the Earth. The annotated genome of Synechococcus sp. PCC 7002, as an ideal model cyanobacterium, is available. A series of transcriptomic and proteomic studies of Synechococcus sp. PCC 7002 cells grown under different conditions have been reported. However, no database of such integrated omics studies has been constructed. Here we present CyanOmics, a database based on the results of Synechococcus sp.
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Convenient and precise strategy for mapping N-glycosylation sites using microwave-assisted acid hydrolysis and characteristic ions recognition
Analytical Chemistry. 2015. Ma, C et al. Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30303 USA.
ABSTRACT: N-glycosylation is one of the most prevalence protein post-translational modifications (PTM) which is involved in several biological processes. Alternation of N-glycosylation is associated with cellular malfunction and development of disease. Thus, investigation of protein N-glycosylation is crucial for diagnosis and treatment of disease. Currently, deglycosylation with peptide N-glycosidase F is the most commonly used technique in N-glycosylation analysis. Additionally, a common error in N-glycosylation site identification, resulting from protein chemical deamidation, has largely been ignored.
[more...]
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Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation
eLife. 2015. Pleiner, T et al. Max Planck Inst Biophys Chem, Dept Cellular Logist, D-37077 Gottingen, Germany.
ABSTRACT: Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems.
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The architecture of a eukaryotic replisome
nature structural & molecular biology. 2015. Sun, JC et al. Rockefeller Univ, DNA Replicat Lab, 1230 York Ave, New York, NY 10021 USA.
ABSTRACT: At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol epsilon is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) alpha-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol epsilon, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol a at the top of CMG.
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Kojak: efficient analysis of chemically cross-linked protein complexes
Journal of Proteome Research. 2015. Hoopmann, MR et al. Inst Syst Biol, 401 Terry Ave North, Seattle, WA 98109 USA.
ABSTRACT: Protein chemical cross-linking and mass spectrometry enable the analysis of protein protein interactions and protein topologies;, however, complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with of without heavy isotope labels.
[more...]
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A strategy for dissecting the architectures of native macromolecular assemblies
Nature methods. 2015. Shi, Y et al. Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, 1230 York Ave, New York, NY 10021 USA.
ABSTRACT: It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
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Conformational states of the full-length glucagon receptor
Nature Communications. 2015. Yang, LL et al. Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Drug Discovery & Design Ctr, 555 Zuchongzhi Rd, Shanghai 201203, Peoples R China.
ABSTRACT: Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain.
[more...]
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ESCRTs cooperate with a selective autophagy receptor to mediate vacuolar targeting of soluble cargos
Molecular cell. 2015. Liu, XM et al. Natl Inst Biol Sci, Collaborat Innovat Ctr Canc Med, Beijing 102206, Peoples R China.
ABSTRACT: Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy.
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Analysis of the histone H3. 1 interactome: a suitable chaperone for the right event
Molecular cell. 2015. Campos, EI et al. NYU, Howard Hughes Med Inst, Sch Med, New York, NY 10016 USA.
ABSTRACT: Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells.
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Structural and functional characterization of CRM1-Nup214 interactions reveals multiple FG-binding sites involved in nuclear export
Cell Reports. 2015. Port, SA et al. Univ Gottingen, GZMB, Inst Microbiol & Genet, Dept Mol Struct Biol, Justus von Liebig Weg 11, D-37077 Gottingen, Germany.
ABSTRACT: CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 angstrom resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor.
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The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes
nature structural & molecular biology. 2015. De, I et al. Max Planck Inst Biophys Chem, D-37077 Gottingen, Germany.
ABSTRACT: Aquarius is a multifunctional putative RNA helicase that binds precursor-mRNA introns at a defined position. Here we report the crystal structure of human Aquarius, revealing a central RNA helicase core and several unique accessory domains, including an ARM-repeat domain. We show that Aquarius is integrated into spliceosomes as part of a pentameric intron-binding complex (IBC) that, together with the ARM domain, cross-links to U2 snRNP proteins within activated spliceosomes; this suggests that the latter aid in positioning Aquarius on the intron.
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The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation
GENES & DEVELOPMENT. 2015. Absmeier, E et al. Free Univ Berlin, Lab Struct Biochem, D-14195 Berlin, Germany.
ABSTRACT: The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an similar to 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes.
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Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex
Journal of Biological chemistry. 2015. Setiaputra, D et al. Univ British Columbia, Dept Biochem & Mol Biol, 2350 Hlth Sci Mall, Vancouver, BC V6T 1Z3, Canada.
ABSTRACT: The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these.
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Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms
AUTOPHAGY. 2015. Chew, LH et al. Univ British Columbia, Dept Biochem & Mol Biol, Vancouver, BC V5Z 1M9, Canada.
ABSTRACT: The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex.
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Comprehensive cross-linking mass spectrometry reveals parallel orientation and flexible conformations of plant HOP2–MND1
Journal of proteome research. 2015. Rampler, E et al. Univ Vienna, Dept Chromosome Biol, Dr Bohr Gasse 9, A-1030 Vienna, Austria.
ABSTRACT: The HOP2-MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2-MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL MS). The final XL MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC-MS/MS detection prior to data evaluation.
[more...]
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The activity of protein phosphatase 5 towards native clients is modulated by the middle-and C-terminal domains of Hsp90
Scientific reports. 2015. Haslbeck, V et al. Tech Univ Munich, Dept Chem, Ctr Integrated Prot Sci Munich, Lichtenbergstr 4, D-85748 Garching, Germany.
ABSTRACT: Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes.
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Assembly and molecular architecture of the phosphoinositide 3-kinase p85α homodimer
Journal of Biological chemistry. 2015. LoPiccolo, J et al. Albert Einstein Coll Med, Dept Biochem, 1300 Morris Pk Ave, Bronx, NY 10461 USA.
ABSTRACT: Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85 alpha, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85 alpha dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85 alpha that is functionally equivalent to native p85 alpha.
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Visualizing the ensemble structures of protein complexes using chemical cross-linking coupled with mass spectrometry
Biophysics reports. 2015. Gong, Zhou et al. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China
ABSTRACT: GRAPHICAL ABSTRACT: ABSTRACT: Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-mol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure.
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Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex
GENES & DEVELOPMENT. 2015. Kapoor, P et al. Univ Texas MD Anderson Canc Ctr, Dept Epigenet & Mol Carcinogenesis, Smithville, TX 78957 USA.
ABSTRACT: ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity.
[more...]
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Chemical cross‐linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex
Protein Science. 2015. Nguyen-Huynh, NT et al. Univ Strasbourg Chim Matiere Complexe, LSMIS, UMR CNRS 7140, 1 Rue Blaise Pascal, F-67008 Strasbourg, France.
ABSTRACT: Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex.
[more...]
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Evidence for two distinct binding sites for lipoprotein lipase on glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)
Journal of Biological chemistry. 2015. Reimund, M et al. Akad Tee 15, EE-12618 Tallinn, Estonia.
ABSTRACT: GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P.
[more...]
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Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
Nucleic Acids Research. 2015. Trahan, C et al. Inst Rech Clin Montreal, Dept Syst Biol, Montreal, PQ H2W 1R7, Canada.
ABSTRACT: Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs.
[more...]
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Probing structures of large protein complexes using zero-length cross-linking
Methods. 2015. Rivera-Santiago, RF et al. Wistar Inst Anat & Biol, 3601 Spruce St, Philadelphia, PA 19104 USA.
ABSTRACT: Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein-protein interactions or the derived distance constraints can be used to verify and refine molecular models. This review focuses on recent advances of "zero-length" cross-linking.
[more...]
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xIP-MS: topological analysis of chromatin-associated protein complexes using single affinity purification
Molecular & Cellular Proteomics. 2015. Matthew M Makowski et al. Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands
ABSTRACT: In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis.
[more...]
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Structure of a yeast spliceosome at 3.6-angstrom resolution
Science. 2015. Yan, CY et al. Tsinghua Univ, Minist Educ, Key Lab Prot Sci,Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci,Ctr Struct Bio, Beijing 100084, Peoples R China.
ABSTRACT: Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat.
[more...]
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Extracting accurate precursor information for tandem mass spectra by RawConverter
Analytical Chemistry. 2015. He, L et al. Scripps Res Inst, Dept Physiol Chem, 10550 North Torrey Pines Rd, La Jolla, CA 92037 USA.
ABSTRACT: Extraction of data from the proprietary RAW files generated by Thermo Fisher mass spectrometers is the primary step for subsequent data analysis. High resolution and high mass accuracy data obtained by state-of-the-art mass spectrometers (e.g., Orbitraps) can significantly improve both peptide/protein identification and quantification. We developed RawConverter, a stand-alone software tool, to improve data extraction on RAW files from high-resolution Thermo Fisher mass spectrometers. RawConverter extracts full scan and MS n data from RAW files like its predecessor RawXtract; most importantly, it associates the accurate precursor mass-tocharge (m/z) value with the tandem mass spectrum.
[more...]
Use: pXtract; pParse



EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra*[S]
Molecular & Cellular Proteomics. 2015. Yuan, ZF et al. Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, Epigenet Program, Room 9-124,3400 Civ Ctr Blvd,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT: Histone post-translational modifications contribute to chromatin function through their chemical properties which influence chromatin structure and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis because of the high sensitivity and the high mass accuracy of this approach that provides confident identification.
[more...]
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Subset of kappa and lambda germline sequences result in light chains with a higher molecular mass phenotype
Journal of Proteome Research. 2015. Barnidge, DR et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT: In our previous work, we showed that electrospray ionization of intact polydonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da.
[more...]
Use: pNovo



A framework of de novo peptide sequencing for multiple tandem mass spectra
IEEE Transactions on Nanobioscience. 2015. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: With tandem mass spectrometry (MS/MS), spectra can be generated by various fragmentation techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD) and so on. At the same time, de novo sequencing using multiple spectra from the same peptide generated by different fragmentation techniques is becoming popular in proteomics studies. The focus of this study is the use of paired spectra from CID (or HCD) and ECD (or ETD) fragmentation because of the complementarity between them.
[more...]
Use: pNovo



Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype
Journal of Proteome Research. 2015. Barnidge, DR et al. Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA.
ABSTRACT: In our previous work, we showed that electrospray ionization of intact polydonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da.
[more...]
Use: pNovo



A Framework of De Novo Peptide Sequencing for Multiple Tandem Mass Spectra
IEEE Transactions on Nanobioscience. 2015. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: With tandem mass spectrometry (MS/MS), spectra can be generated by various fragmentation techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD) and so on. At the same time, de novo sequencing using multiple spectra from the same peptide generated by different fragmentation techniques is becoming popular in proteomics studies. The focus of this study is the use of paired spectra from CID (or HCD) and ECD (or ETD) fragmentation because of the complementarity between them.
[more...]
Use: pNovo



Pulsed azidohomoalanine labeling in mammals (PALM) detects changes in liver-specific LKB1 knockout mice
Journal of proteome research. 2015. McClatchy, DB et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Mol Biol, 10550 North Torrey Pines Rd, La Jolla, CA 92037 USA.
ABSTRACT: Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the non-canonical amino acid, azidohomoalanine (AHA).
[more...]
Use: pQuant



Profiling the RNA editomes of wild-type C. elegans and ADAR mutants
Genome research. 2015. Zhao, HQ et al. Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT: RNA editing increases transcriptome diversity through post-transcriptional modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in higher eukaryotes. Caenorhabditis elegans has two ADARs, ADR-1 and ADR-2, but their functions remain unclear. Here, we profiled the RNA editomes of C. elegans at different developmental stages of wild-type and ADAR mutants. We developed a new computational pipeline with a "bisulfite-seq-mapping-like" step and achieved a threefold increase in identification sensitivity.
[more...]
Use: pQuant



Structure of the voltage-gated calcium channel Cav1.1 complex
SCIENCE. 2015. et al. Tsinghua Univ, State Key Lab Membrane Biol, Beijing 100084, Peoples R China
ABSTRACT: Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning.
[more...]
Use: pLink




2014




UvrD facilitates DNA repair by pulling RNA polymerase backwards
Nature. 2014. Epshtein, V et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT: UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes.
[more...]
Use: pLink; pLabel; pFind



1-Dodecyl-3-methylimidazolium chloride-assisted sample preparation method for efficient integral membrane proteome analysis
Analytical Chemistry. 2014. Zhao, Q et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT: Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs.
[more...]
Use: pXtract; pBuild; pFind



N-glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry
Journal of proteome research. 2014. Cao, Liwei et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo H-f), respectively, prior to LC-MS/MS analysis.
[more...]
Use: pFind; pXtract; pBuild



Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis
Molecular & Cellular Proteomics. 2014. Liu, FY et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT: The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen.
[more...]
Use: pFind



Transferred subgroup false discovery rate for rare post-translational modifications detected by mass spectrometry
Molecular & Cellular Proteomics. 2014. Fu, Y et al. 55 Zhongguancun East Rd, Beijing 100190, Peoples R China.
ABSTRACT: In shotgun proteomics, high-throughput mass spectrometry experiments and the subsequent data analysis produce thousands to millions of hypothetical peptide identifications. The common way to estimate the false discovery rate (FDR) of peptide identifications is the target-decoy database search strategy, which is efficient and accurate for large datasets. However, the legitimacy of the target-decoy strategy for protein-modification-centric studies has rarely been rigorously validated. It is often the case that a global FDR is estimated for all peptide identifications including both modified and unmodified peptides, but that only a subgroup of identifications with a certain type of modification is focused on.
[more...]
Use: pFind



Evaluation of proteomic search engines for the analysis of histone modifications
Journal of Proteome Research. 2014. Yuan, ZF et al. Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, 3400 Civ Ctr,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT: Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results.
[more...]
Use: pFind; pParse



Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass …
Analytical Chemistry. 2014. Cao, QC et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated alpha-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS3 scanning under collision-induced dissociation (CID) by ion trap mass spectrometry.
[more...]
Use: pFind



Various conotoxin diversifications revealed by a venomic study of Conus flavidus
Molecular & Cellular Proteomics. 2014. Lu, AP et al. Tongji Univ, Sch Life Sci & Technol, Inst Prot Res, 1239 Siping Rd, Shanghai 200092, Peoples R China.
ABSTRACT: Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C.
[more...]
Use: pXtract; pFind; pLabel



Phosphoproteomic Analysis Provides Novel Insights into Stress Responses in Phaeodactylum tricornutum, a Model Diatom
Journal of Proteome Research. 2014. Chen, Z et al. Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China.
ABSTRACT: Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P.
[more...]
Use: pFind; pBuild



A precise approach in large scale core-fucosylated glycoprotein identification with low-and high-normalized collision energy
Journal of Proteomics. 2014. Ma, C et al. Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30302 USA.
ABSTRACT: The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications. However, due to the complex fragmentation pattern of CF-glycopeptides, accurately identifying CF-glycosylation sites via mass spectrometry with high throughput remains a formidable challenge.
[more...]
Use: pFind; pBuild



In vivo proximity labeling for the detection of protein–protein and protein–RNA interactions
Journal of Proteome Research. 2014. Beck, DB et al. Univ Penn, Perelman Sch Med, Epigenet Program, Dept Cell & Dev Biol, Philadelphia, PA 19103 USA.
ABSTRACT: Accurate and sensitive detection of protein-protein and protein-RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo.
[more...]
Use: pFind; pParse



Integration of mass spectrometry and RNA‐Seq data to confirm human ab initio predicted genes and lncRNAs
Proteomics. 2014. Sun, Han et al. Shanghai Acad Sci & Technol, Shanghai Ctr Bioinformat Technol, Shanghai 201203, Peoples R China
ABSTRACT: MS/MS has been used to improve genome annotation in various organisms. The classical approach is to construct comprehensive theoretical peptide database with six frame translation model from the whole ORF of a genome and search against this database with real MS/MS spectra. In this work we took a more focused approach, we constructed a database containing only peptides from the ab initio predicted genes from current human genome annotation, and all theoretical peptides from currently annotated lncRNAs, and searched such a database with MS/MS data from human Hela cell line.
[more...]
Use: pFind



Accelerating the scoring module of mass spectrometry-based peptide identification using GPUs
BMC Bioinformatics. 2014. Li, Y et al. Hong Kong Baptist Univ, Dept Comp Sci, Kowloon Tong, Hong Kong, Peoples R China.
ABSTRACT: Background: Tandem mass spectrometry-based database searching is currently the main method for protein identification in shotgun proteomics. The explosive growth of protein and peptide databases, which is a result of genome translations, enzymatic digestions, and post-translational modifications (PTMs), is making computational efficiency in database searching a serious challenge. Profile analysis shows that most search engines spend 50%-90% of their total time on the scoring module, and that the spectrum dot product (SDP) based scoring module is the most widely used.
[more...]
Use: pFind



Complex proteomes analysis using label-free mass spectrometry-based quantitative approach coupled with biomedical knowledge
International Conference on Intelligent Information Processing. 2014. Chao Pan et al. College of Biomedical Engineering and Instrument Science, Key Laboratory of Biomedical Engineering of Ministry of Education of China, Zhejiang University
ABSTRACT: Label-free quantitative proteomics based on mass spectrometry plays an essential role in large-scale analysis of complex proteomes. Meanwhile, quantitative proteomics is not only a way for data processing, but also an important approach for exploring protein functions and interactions in a large-scale manner. An effective method combining quantitation and qualification should be built. To systematically overcome this challenge, we proposed a new label-free quantitative method using spectral counting in the proposed method, the count of shared peptides was considered as an optimized factor to accurately appraise abundance of Isoforms for complex proteomes.
[more...]
Use: pFind



N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based Enrichment, Parallel …
Journal of proteome research. 2014. Cao, Liwei et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT: N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo H-f), respectively, prior to LC-MS/MS analysis.
[more...]
Use: pFind; pXtract; pBuild



蛋白质序列及其翻译后修饰鉴定的盲搜索算法评价
科研信息化技术与应用. 2014. 黄潘育 et al. 中国科学院计算机网络信息中心超级计算中心
ABSTRACT: 蛋白质翻译后修饰鉴定是计算蛋白质科学的重要研究方向,研发速度快和精度高的鉴定算法及软件,是该方向的一个主要问题。本文回顾了蛋白质翻译后修饰鉴定的各类算法,并主要对蛋白质序列及其翻译后修饰鉴定的盲搜索算法和软件进行研究。通过对具有代表性的两个盲搜索软件InsPecT和SIMS,以及限定性搜索软件pFind的对比分析,对盲搜索算法在蛋白质序列及其翻译后修饰方面的瓶颈、问题及未来发展趋势做出了评价
Use: pFind



Proteome informatics research group (iPRG) \_2012: a study on detecting modified peptides in a complex mixture
Molecular & Cellular Proteomics. 2014. Chalkley, RJ et al. 600 16th St,Genentech Hall,Room N474A, San Francisco, CA 94158 USA.
ABSTRACT: The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed.
[more...]
Use: pFind



Proteogenomic analysis and global discovery of posttranslational modifications in prokaryotes
Proceedings of the National Academy of Sciences of the United States of America. 2014. Yang, MK et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT: We describe an integrated workflow for proteogenomic analysis and global profiling of posttranslational modifications (PTMs) in prokaryotes and use the model cyanobacterium Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) as a test case. We found more than 20 different kinds of PTMs, and a holistic view of PTM events in this organism grown under different conditions was obtained without specific enrichment strategies. Among 3,186 predicted protein-coding genes, 2,938 gene products (> 92%) were identified.
[more...]
Use: pFind



Phosphoproteomic analysis provides novel insights into stress responses in Phaeodactylum tricornutum, a model diatom
Journal of Proteome Research. 2014. Chen, Z et al. Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China.
ABSTRACT: Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P.
[more...]
Use: pFind; pBuild



Identification of human tissue kallikrein 6 as a potential marker of laryngeal cancer based on the relevant secretory/releasing protein database
Disease Markers. 2014. Zhang, Y et al. Peking Union Med Coll, Canc Inst Hosp, Beijing Key Lab Carcinogenesis & Canc Prevent, State Key Lab Mol Oncol, POB 2258, Beijing 100021, Peoples R China.
ABSTRACT: This study was aimed to create a large-scale laryngeal cancer relevant secretory/releasing protein database and further discover candidate biomarkers. Methods. Primary tissue cultures were established using tumor tissues and matched normal mucosal tissues collected from four laryngeal cancer patients. Serum-free conditioned medium( CM) samples were collected. These samples were then sequentially processed by SDS-PAGE separation, trypsin digestion, and LC-MS/MS analysis. The candidates in the database were validated by ELISA using plasma samples from laryngeal cancer patients, benign patients, and healthy individuals.
[more...]
Use: pFind



Complex Proteomes Analysis Using Label-Free Mass Spectrometry-Based Quantitative Approach Coupled with Biomedical Knowledge
International Conference on Intelligent Information Processing. 2014. Chao Pan et al. College of Biomedical Engineering and Instrument Science, Key Laboratory of Biomedical Engineering of Ministry of Education of China, Zhejiang University
ABSTRACT: Label-free quantitative proteomics based on mass spectrometry plays an essential role in large-scale analysis of complex proteomes. Meanwhile, quantitative proteomics is not only a way for data processing, but also an important approach for exploring protein functions and interactions in a large-scale manner. An effective method combining quantitation and qualification should be built. To systematically overcome this challenge, we proposed a new label-free quantitative method using spectral counting in the proposed method, the count of shared peptides was considered as an optimized factor to accurately appraise abundance of Isoforms for complex proteomes.
[more...]
Use: pFind



Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry
Analytical Chemistry. 2014. Cao, QC et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated alpha-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS3 scanning under collision-induced dissociation (CID) by ion trap mass spectrometry.
[more...]
Use: pFind



Visualization of arrestin recruitment by a G-protein-coupled receptor
Nature. 2014. Shukla, AK et al. Duke Univ, Med Ctr, Dept Med, Durham, NC 27710 USA.
ABSTRACT: G-protein-coupled receptors (GPCRs) are critically regulated by beta-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling(1-5). A recent surge of structural data on a number of GPCRs, including the beta(2) adrenergic receptor (beta(2)AR)-G-proteincomplex, has provided novel insights into the structural basis of receptor activation(6-11). However, complementary information has been lacking on the recruitment of beta-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-beta-arrestin complexes for structural studies.
[more...]
Use: pLink



Structural characterization by cross-linking reveals the detailed architecture of a coatomer-related heptameric module from the nuclear pore complex
Molecular & Cellular Proteomics. 2014. Shi, Y et al. Rockefeller Univ, Lab Mass Spectrometry & Gaseous Ion Chem, Box 170, New York, NY 10065 USA.
ABSTRACT: Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout. In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents.
[more...]
Use: pXtract; pLink



Matching cross-linked peptide spectra: only as good as the worse identification
Molecular & Cellular Proteomics. 2014. Trnka, MJ et al. 600 16th St,Genentech Hall,Room N474A, San Francisco, CA 94158 USA.
ABSTRACT: Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders.
[more...]
Use: pLink



Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit
nature structural & molecular biology. 2014. Cevher, MA et al. Rockefeller Univ, Lab Biochem & Mol Biol, 1230 York Ave, New York, NY 10021 USA.
ABSTRACT: The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II.
[more...]
Use: pLink



Architecture of the Saccharomyces cerevisiae SAGA transcription coactivator complex
EMBO. 2014. Yan Han et al. Institute for Systems Biology, Seattle, WA, USA
ABSTRACT: The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation (HAT) and deubiquitination (DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA-TBP complex. We find that the SAGA Taf and Taf-like subunits form a TFIID-like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules.
[more...]
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Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway
Molecular & Cellular Proteomics . 2014. Algret, R et al. Univ Paris 11, CNRS UMR 8126, Inst Gustave Roussy, 11 Rue Edouard Vaillant, F-94805 Villejuif, France.
ABSTRACT: The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex comprises two regions that are structurally and functionally distinct.
[more...]
Use: pLink



Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex
Nature Structural & Molecular Biology. 2014. Knutson, BA et al. Fred Hutchinson Canc Res Ctr, Div Basic Sci, 1124 Columbia St, Seattle, WA 98104 USA.
ABSTRACT: Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Poll factor SL1.
[more...]
Use: pLink



A mechanism for actin filament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface
Journal of Biological chemistry. 2014. Wong, W et al. Imperial Coll Sci Technol & Med, Dept Life Sci, London SW7 2AZ, England.
ABSTRACT: Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain.
[more...]
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In Vitro Reconstitution of Yeast tUTP/UTP A and UTP B Subcomplexes Provides New Insights into Their Modular Architecture
PLOS ONE. 2014. Poll, G et al. Univ Regensburg, Lehrstuhl Biochem 3, D-93053 Regensburg, Germany.
ABSTRACT: Eukaryotic ribosome biogenesis is a multistep process involving more than 150 biogenesis factors, which interact transiently with pre-ribosomal particles to promote their maturation. Some of these auxiliary proteins have been isolated in complexes found separate from the ribosomal environment. Among them, are 3 large UTP subcomplexes containing 6 or 7 protein subunits which are involved in the early steps of ribosome biogenesis. The composition of the UTP subcomplexes and the network of binary interactions between protein subunits have been analyzed previously.
[more...]
Use: pLink



Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2
PLOS Biology. 2014. Zhang, JM et al. Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT: Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16(XPF)-Swi10(ERCC1) and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 39 endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 59 endonuclease activity of Dna2.
[more...]
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Identification of protein partners in mycobacteria using a single-step affinity purification method
PLOS ONE. 2014. Plocinski, P et al. Polish Acad Sci, Inst Biochem & Biophys, Warsaw, Poland.
ABSTRACT: Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium-and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli.
[more...]
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Combinatorial approach for large-scale identification of linked peptides from tandem mass spectrometry spectra
Molecular & Cellular Proteomics. 2014. Wang, J et al. Univ Calif San Diego, Ctr Computat Mass Spectrometry, Dept Comp Sci & Engn, 9500 Gilman Dr,Mail Code 0404, La Jolla, CA 92093 USA.
ABSTRACT: The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides.
[more...]
Use: pLink



Characterization of the Raptor/4E-BP1 interaction by chemical cross-linking coupled with mass spectrometry analysis
Journal of Biological chemistry. 2014. Coffman, K et al. Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT: Background: mTORC1 recruits its substrate 4E-BP1 via Raptor/4E-BP1 interaction. Chemical cross-linking/mass spectrometry permits characterization of protein-protein interactions. Results: Cross-linked peptides between Raptor and 4E-BP1 were identified. Raptor intramolecular cross-links were also identified. Conclusion: Raptor N-terminal region containing RNC1 is implicated in the interaction with the central region of 4E-BP1. Significance: Our study provides novel insight into how mTORC1 recognizes 4E-BP1.
[more...]
Use: pLink



Enhanced identification of zero-length chemical cross-links using label-free quantitation and high-resolution fragment ion spectra
Journal of Proteome Research. 2014. Sriswasdi, S et al. Wistar Inst Anat & Biol, Ctr Syst & Computat Biol, 3601 Spruce St, Philadelphia, PA 19104 USA.
ABSTRACT: Chemical cross-linking coupled to mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. "Zero-length" cross-links have greater value for these applications than longer cross-links because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification.
[more...]
Use: pLink



Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass …
Journal of Proteomics. 2014. Tiroli-Cepeda, AO et al. Univ Estadual Campinas, UNICAMP, Inst Chem, POB 6154, BR-13083970 Campinas, SP, Brazil.
ABSTRACT: Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp.
[more...]
Use: pLink



Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA
Biochmical and Biophysical Research Communications. 2014. Shapiro, AB et al. AstraZeneca R&D Boston, 35 Gatehouse Dr, Waltham, MA 02451 USA.
ABSTRACT: LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichia coli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo.
[more...]
Use: pLink; pLabel



Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity
PLOS ONE. 2014. Plocinski, P et al. Polish Acad Sci, Inst Biochem & Biophys, Warsaw, Poland.
ABSTRACT: Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium-and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli.
[more...]
Use: pLink



Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays
Journal of Proteomics. 2014. Tiroli-Cepeda, AO et al. Univ Estadual Campinas, UNICAMP, Inst Chem, POB 6154, BR-13083970 Campinas, SP, Brazil.
ABSTRACT: Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp.
[more...]
Use: pLink



Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1$\alpha$ transactivation
BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS. 2014. Zhao, L et al. Beijing Inst Radiat Med, 27 Taiping Rd, Beijing 100850, Peoples R China.
ABSTRACT: Hepatocyte nuclear factor-1 alpha (HNF1 alpha) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1 alpha. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1 alpha at Ser249. We also found that the ATM protein kinase phosphorylated HNF1 alpha at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1 alpha at Ser249 in vivo.
[more...]
Use: pXtract



De novo identification and quantification of single amino-acid variants in human brain
Journal of Molecular Cell Biology. 2014. Su, ZD et al. Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, Key Lab Syst Biol, Shanghai 200031, Peoples R China.
ABSTRACT: The detection of single amino-acid variants (SAVs) usually depends on single-nucleotide polymorphisms (SNPs) database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate. In human brain tissues, we can confidently identify known and novel protein variants with diverse origins.
[more...]
Use: pNovo



NovoHCD: de novo peptide sequencing from HCD spectra
IEEE Transactions on Nanobioscience. 2014. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: In recent years, de novo peptide sequencing from mass spectrometry data has developed as one of the major peptide identification methods with the emergence of new instruments and advanced computational methods. However, there are still limitations to this method; for example, the typically used spectrum graph model cannot represent all the information and relationships inherent in tandem mass spectra (MS/MS spectra). Here, we present a new method named NovoHCD which applies a spectrum graph model with multiple types of edges (called a multi-edge graph), and integrates into it amino acid combination (AAC) information and peptide tags.
[more...]
Use: pNovo



NovoExD: De novo peptide sequencing for ETD/ECD spectra
IEEE-ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS. 2014. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: De novo peptide sequencing using tandem mass spectrometry (MS/MS) data has become a major computational method for sequence identification in recent years. With the development of new instruments and technology, novel computational methods have emerged with enhanced performance. However, there are only a few methods focusing on ECD/ETD spectra, which mainly contain variants of c-ions and z-ions. Here, a de novo sequencing method for ECD/ETD spectra, NovoExD, is presented. NovoExD applies a new form of spectrum graph with multiple edge types (called a GMET), considers multiple peptide tags, and integrates amino acid combination (AAC) and fragment ion charge information.
[more...]
Use: pNovo



NovoHCD: De novo Peptide Sequencing From HCD Spectra
IEEE Transactions on Nanobioscience. 2014. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: In recent years, de novo peptide sequencing from mass spectrometry data has developed as one of the major peptide identification methods with the emergence of new instruments and advanced computational methods. However, there are still limitations to this method; for example, the typically used spectrum graph model cannot represent all the information and relationships inherent in tandem mass spectra (MS/MS spectra). Here, we present a new method named NovoHCD which applies a spectrum graph model with multiple types of edges (called a multi-edge graph), and integrates into it amino acid combination (AAC) information and peptide tags.
[more...]
Use: pNovo



Hunting for unexpected post-translational modifications by spectral library searching with tier-wise scoring
Journal of Proteome Research. 2014. Ma, CWM et al. Hong Kong Univ Sci & Technol, Div Biomed Engn, Hong Kong, Hong Kong, Peoples R China.
ABSTRACT: Discovering novel post-translational modifications (PTMS) to proteins and detecting specific modification sites on proteins is one of the last frontiers of proteomics. At present, hunting for post-translational modifications remains challenging in widely practiced shotgun proteomics workflows due to the typically low abundance of modified peptides and the greatly inflated search space as more potential mass shifts are considered by the search engines. Moreover, most popular search methods require that the user specifies the modification(s) for which to search; therefore, unexpected and novel PTMs will not be detected.
[more...]
Use: pMatch




2013 or earlier




Large-scale global identification of protein lysine methylation in vivo
Epigenetics. 2013. Cao, Xing-Jun et al. Univ Penn, Dept Biochem & Biophys, Perelman Sch Med, Epigenet Program, 1009C Stellar Chance Labs, Philadelphia, PA 19104 USA
ABSTRACT: Lysine methylation mediated by methyltransferase enzymes is present on multiple proteins throughout the cell; however, methods to uncover and characterize global protein lysine methylation patterns do not readily exist. Here we developed pan-specific methyl lysine antibodies that we utilized in immunoprecipitation experiments coupled with mass spectrometry to yield one of the first large-scale surveys of protein lysine methylation in vivo. In total, 552 different lysine methylation sites were determined, making this one of the most comprehensive global studies published to date.
[more...]
Use: pFind



A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue
Journal of Chromatography A. 2012. Jiang, H et al. 457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix.
[more...]
Use: pFind; pXtract



Biphasic microreactor for efficient membrane protein pretreatment with a combination of formic acid assisted solubilization, on-column pH adjustment, reduction, alkylation, and tryptic digestion
Analytical chemistry. 2013. Zhao, Qun et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China
ABSTRACT: Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column.
[more...]
Use: pFind; pXtract; pBuild



Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment
Analytical and Bioanalytical Chemistry. 2013. Zhao, XY et al. Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT: Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions.
[more...]
Use: pFind



NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free proteome quantification
Analyst. 2012. Wu, Q et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China.
ABSTRACT: Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies.
[more...]
Use: pFind; pXtract; pBuild



Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp …
Journal of proteome research. 2013. Yang, MK et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT: Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues.
[more...]
Use: pFind



c-Abl promotes osteoblast expansion by differentially regulating canonical and non-canonical BMP pathways and p16INK4a expression
Nature cell biology. 2012. Kua, Hui-Yi et al. Shanghai Jiao Tong Univ, Minist Educ, Bio X Inst, Key Lab Genet Dev & Neuropsychiat Disorders, Shanghai 200240, Peoples R China
ABSTRACT: Defects in stem cell renewal or progenitor cell expansion underlie ageing-related diseases such as osteoporosis. Yet much remains unclear about the mechanisms regulating progenitor expansion. Here we show that the tyrosine kinase c-Abl plays an important role in osteoprogenitor expansion. c-Abl interacts with and phosphorylates BMPRIA and the phosphorylation differentially influences the interaction of BMPRIA with BMPRII and the Tab1-Tak1 complex, leading to uneven activation of Smad1/5/8 and Erk1/2, the canonical and non-canonical BMP pathways that direct the expression of p(16INK4a).
[more...]
Use: pFind



Mass defect-based pseudo-isobaric dimethyl labeling for proteome quantification
Analytical Chemistry. 2013. Zhou, Y et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian, Peoples R China.
ABSTRACT: Discovering differentially expressed proteins in various biological samples requires proteome quantification methods with accuracy, precision, and wide dynamic range. This study describes a mass defect-based pseudo-isobaric dimethyl labeling (pIDL) method based on the subtle mass defect differences between C-12/C-13 and H-1/H-2. Lys-C protein digests were labeled with CD2O/(CD2O)-C-13 and reduced with NaCNBD3/NaCNBH3 as heavy and light isotopologues, respectively. The fragment ion pairs with mass differences of 5.84 mDa were resolved by high-resolution tandem mass spectrometry (MS/MS) and used for quantification.
[more...]
Use: pXtract; pFind



The ovarian cancer‐derived secretory/releasing proteome: A repertoire of tumor markers
Proteomics. 2012. Zhang, Y et al. Dept Etiol & Carcinogenesis, POB 2258, Beijing 100021, Peoples R China.
ABSTRACT: Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery. First, primary organ cultures were established with the tumor tissues and corresponding normal tissues obtained from six ovarian cancer patients, and the serum-free conditioned medium (CM) samples were collected for proteomic analysis.
[more...]
Use: pFind



Integrated platform for proteome profiling with combination of microreversed phase based protein and peptide separation via online solvent exchange and protein …
Analytical chemistry. 2012. Yuan, Huiming et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Key Lab Separat Sci Analyt Chem, Dalian Inst Chem Phys, Dalian 116023, Peoples R China
ABSTRACT: An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (mu RPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by mu RPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (mu RPLC-ESI-MS/MS). To evaluate the performance of such a platform, a three-protein mixture with mass ranging from 5 to 500 ng was analyzed automatically.
[more...]
Use: pFind; pXtract; pBuild



Determination of monoisotopic masses of chimera spectra from high-resolution mass spectrometric data by use of isotopic peak intensity ratio modeling
Rapid Communications in Mass Spectrometry. 2012. Niu, Ming et al. Natl Engn Res Ctr Prot Drugs, Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, 33 Life Sci Pk Rd, Beijing 102206, Peoples R China
ABSTRACT: RATIONALE Chimera spectra make it challenging to identify proteins in complex mixtures by LC/MS/MS. Approximately half of the spectra collected are chimera spectra even when high-resolution tandem mass spectrometry is used. Chimera spectra are generated from the co-fragmentation of different co-elute peptides, and it is often difficult to distinguish monoisotopic precursors of these peptides from each other. METHODS In this paper, we propose a peak intensity ratio-based monoisotopic peak determination algorithm (PIRMD) to distinguish different monoisotopic precursors of chimera spectra.
[more...]
Use: pFind; pBuild; pParse; pLabel



Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris
Biotechnology Letters. 2013. Lei, D et al. Nanchang Univ, State Key Lab Food Sci & Technol, Nanjing East Rd 235, Nanchang 330047, Jiangxi, Peoples R China.
ABSTRACT: Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %.
[more...]
Use: pFind



Method for rapid protein identification in a large database
Biomed Research International. 2013. Wenli Zhang et al. Institute of Computing Technology, Chinese Academy of Sciences
ABSTRACT: Protein identification is an integral part of proteomics research. The available tools to identify proteins in tandem mass spectrometry experiments are not optimized to face current challenges in terms of identification scale and speed owing to the exponential growth of the protein database and the accelerated generation of mass spectrometry data, as well as the demand for nonspecific digestion and post-modifications in complex-sample identification. As a result, a rapid method is required to mitigate such complexity and computation challenges.
[more...]
Use: pFind



Speeding up scoring module of mass spectrometry based protein identification by GPU
2012 IEEE 14TH INTERNATIONAL CONFERENCE ON HIGH PERFORMANCE COMPUTING AND COMMUNICATIONS & 2012 IEEE 9TH INTERNATIONAL CONFERENCE ON EMBEDDED SOFTWARE AND SYSTEMS (HPCC-ICESS). 2012. Li, Y et al. Hong Kong Baptist Univ, Dept Comp Sci, Kowloon Tong, Hong Kong, Peoples R China.
ABSTRACT: Database searching is a main method for protein identification in shotgun proteomics, and many research efforts are dedicated to improving its effectiveness. However, the efficiency of database searching is facing a serious challenge, due to the ever fast growth of protein and peptide databases resulted from genome translations, enzymatic digestions, and post-translational modifications (PTMs). On the other hand, as a general-purpose and high performance parallel hardware, Graphics Processing Units (GPUs) develop continuously and provide another promising platform for parallelizing database searching based protein identification.
[more...]
Use: pFind



Efficient discovery of abundant post-translational modifications and spectral pairs using peptide mass and retention time differences
BMC bioinformatics. 2009. Fu, Yan et al. Chinese Acad Sci, Inst Comp Technol, Beijing 100190, Peoples R China
ABSTRACT: Background: Peptide identification via tandem mass spectrometry is the basic task of current proteomics research. Due to the complexity of mass spectra, the majority of mass spectra cannot be interpreted at present. The existence of unexpected or unknown protein post-translational modifications is a major reason.Results: This paper describes an efficient and sequence database-independent approach to detecting abundant post-translational modifications in high-accuracy peptide mass spectra. The approach is based on the observation that the spectra of a modified peptide and its unmodified counterpart are correlated with each other in their peptide masses and retention time.
[more...]
Use: pFind



A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide …
Journal of Chromatography A. 2012. Jiang, H et al. 457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix.
[more...]
Use: pFind; pXtract



Biphasic microreactor for efficient membrane protein pretreatment with a combination of formic acid assisted solubilization, on-column pH adjustment, reduction …
Analytical chemistry. 2013. Zhao, Qun et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian 116023, Peoples R China
ABSTRACT: Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column.
[more...]
Use: pFind; pXtract; pBuild



NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free …
Analyst. 2012. Wu, Q et al. Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Natl Chromatog Res & Anal Ctr, Dalian 116023, Peoples R China.
ABSTRACT: Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies.
[more...]
Use: pFind; pXtract; pBuild



带喷头混合型毛细管色谱柱的制备及其在蛋白质组分离分析中的应用
中国科学. B辑, 化学. 2009. 张养军 et al. 军事医学科学院放射与辐射医学研究所,北京蛋白质组研究中心, 蛋白质组学国家重点实验室, 北京 102206, 中国; 沈阳药科大学, 沈阳, 辽宁 110015, 中国
ABSTRACT: A capillary chromatographic column with an emitter at one end is prepared with monolithic material in the emitter as a cribellum for the capillary column by using lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA), and then the other empty part of the capillary column with an emitter is packed with octadecylsilica particles(ODS). The property of monolithic part of the capillary column is similar to that of reversed-phase packing materials packed in the capillary column and benefits the improvement on separation efficiency and prevents the clogging problem of the capillary column in the tapered tip during its use.
[more...]
Use: pFind



原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用
色谱. 2013. 周廉淇 et al. 蛋白质组学国家重点实验室,北京蛋白质组研究中心,军事医学科学院放射与辐射医学研究所
ABSTRACT: The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks such as long incubation time, enzyme autodigestion, and non-reusability. Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor. The digestion efficiency, repeatability and recovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 min.
[more...]
Use: pFind; pBuild



Thioredoxin and thioredoxin reductase control tissue factor activity by thiol redox-dependent mechanism
Journal of Biological chemistry. 2013. Wang, P et al. Univ Chinese Acad Sci, Coll Life Sci, YuQuan Rd 19 A, Beijing 100049, Peoples R China.
ABSTRACT: Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond.
[more...]
Use: pFind



Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma
Science China Life Sciences. 2013. Yang, L et al. Peking Union Med Coll, Canc Inst Hosp, Dept Abdominal Surg, Beijing 100021, Peoples R China.
ABSTRACT: For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ.
[more...]
Use: pFind



N-linked glycoproteome profiling of human serum using tandem enrichment and multiple fraction concatenation
Electrophoresis. 2013. Ma, C et al. Beijing Inst Technol, Sch Life Sci & Technol, Beijing 100081, Peoples R China.
ABSTRACT: N-linked glycosylation is an important protein posttranslational modification that is involved in numerous biological processes. Different methods, including chemical reaction and affinity interaction, have been developed to enrich glycosylated peptides or proteins from biological systems. However, due to the common occurrence of low glycosites occupancy in proteins and the low efficiency of enrichment approaches, only a small fraction of protein glycosites have been reported. In this study, we combined the glycopeptide enrichment strategy for broad analysis of human serum N-glycoproteins using a tandem enrichment method coupling lectin affinity capture with HILIC.
[more...]
Use: pFind



Global phosphoproteomic analysis reveals diverse functions of serine/threonine/tyrosine phosphorylation in the model cyanobacterium Synechococcus sp. strain PCC 7002
Journal of proteome research. 2013. Yang, MK et al. Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT: Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues.
[more...]
Use: pFind



Fragmentation pattern of glycated peptides derived from D-glucose by tandem mass spectrometry
2013 ICME International Conference on Complex Medical Engineering (CME). 2013. Su, R et al. Beijing Inst Technol, Sch Life Sci & Technol, Beijing 100081, Peoples R China.
ABSTRACT: Evidence shows that non-enzymatic glycation plays an important role in the development of diabetes, diabetes-related complications and neurodegenerative diseases. Mass spectrometric methods have been used for non-enzymatic glycation of proteins. At present, CID remains the major fragmentation method for peptide sequencing. In this study, we utilized synthesized peptide models to study fragmentation spectra of glycated peptides, which can be easily studied using ESI-MS. MS/MS fragmentation whose characteristics of glycated peptides will help for large-scale glycated proteomics analyses of complex plasma and tissue samples.
Use: pFind; pBuild; pLabel



Improved proteomic analysis pipeline for LC-ETD-MS/MS using charge enhancing methods
Molecular Biosystems. 2012. Xie, LQ et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT: Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem).
[more...]
Use: pFind



Cross-linking and mass spectrometry methodologies to facilitate structural biology: finding a path through the maze
Journal of Structural and Functional Genomics . 2013. Merkley, Eric D. et al. Biological Sciences Division, Pacific Northwest, National Laboratory
ABSTRACT: Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes.
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Use: pLink



Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction
Skeletal Muscle. 2011. Mate, SE et al. George Washington Univ, Dept Biochem & Mol Genet, IBS, Washington, DC USA.
ABSTRACT: Background: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins.
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Use: pXtract



UniNovo: a universal tool for de novo peptide sequencing
Bioinformatics. 2013. Jeong, K et al. Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92093 USA.
ABSTRACT: Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests.
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Use: pNovo



UniNovo : A Universal Tool for de Novo Peptide Sequencing
BIOINFORMATICS. 2013. Chuang Li et al. Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92093 USA
ABSTRACT: Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests.
[more...]
Use: pNovo



Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)
Proceedings of the National Academy of Sciences of the United States of America. 2012. Zhao, YM et al. Natl Inst Biol Sci, Beijing 102206, Peoples R China.
ABSTRACT: Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to supportmajor spermprotein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition.
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Use: pNovo



Identification of b-/y-ions in MS/MS spectra using a two stage neural network
Proteome Science. 2013. Cleveland, JP et al. Univ S Carolina, Dept Comp Sci, Columbia, SC 29208 USA.
ABSTRACT: Independent of the approach used, the ability to correctly interpret tandem MS data depends on the quality of the original spectra. Even in the case of the highest quality spectra, the majority of spectral peaks can not be reliably interpreted. The accuracy of sequencing algorithms can be improved by filtering out such 'noise' peaks. Preprocessing MS/MS spectra to select informative ion peaks increases accuracy and reduces the processing time. Intuitively, the mix of informative versus non-informative peaks has a direct effect on the quality and size of the resulting candidate peptide search space.
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Use: pNovo



A multi-edge graph based de novo peptide sequencing method for HCD spectra
2013 IEEE INTERNATIONAL CONFERENCE ON BIOINFORMATICS AND BIOMEDICINE (BIBM). 2013. Yan, Y et al. Univ Saskatchewan, Div Biomed Engn, Saskatoon, SK S7N 5A9, Canada.
ABSTRACT: In recent years, de novo peptide sequencing from mass spectrometry data has developed as one of the major peptide identification methods with the emergence of new instruments and advanced computational methods. However, there are still limitations to this method; for example, the typically used spectrum graph model cannot represent all the information and relationships inherent in tandem mass spectra (MS/MS spectra). Here, we present a new spectrum graph model with multiple types of edges (called a multi-edge graph), and integrate amino acid combination (AAC) information and peptide tags into it for peptide sequencing.
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Use: pNovo



UniNovo: a universal tool for de novo peptide sequencing
Bioinformatics. 2013. Jeong, K et al. Univ Calif San Diego, Dept Elect & Comp Engn, San Diego, CA 92093 USA.
ABSTRACT: Motivation: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests.
[more...]
Use: pNovo



Inverse regulation in the metabolic genes pckA and metE revealed by proteomic analysis of the Salmonella RcsCDB regulon
Journal of Proteome Research. 2011. Paradela, A et al. CSIC, Ctr Nacl Biotecnol, Dept Biotecnol Microbiana, Madrid, Spain.
ABSTRACT: The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatography-electrospray ionization tandem mass spectrometry.
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Use: pScan