Molecular & Cellular Proteomics最新文献

{"title":"Addressing NHS Chemistry: Efficient Quenching of Excess TMT Reagent and Reversing TMT Overlabeling in Proteomic Samples by Methylamine.","authors":"Yana Demyanenko, Xintong Sui, Andrew M Giltrap, Benjamin G Davis, Bernhard Kuster, Shabaz Mohammed","doi":"10.1016/j.mcpro.2025.100948","DOIUrl":"10.1016/j.mcpro.2025.100948","url":null,"abstract":"<p><p>N-hydroxysuccinimide (NHS) ester chemistry is used extensively across proteomics sample preparation. One of its increasingly prevalent applications is in isobaric reagent-based quantitation such as isobaric tags for relative and absolute quantitation and tandem mass tag approaches. In these methods, labeling on the primary amines of lysine residues and N termini of tryptic peptides via amide formation (N-derivatives) from corresponding NHS ester reagents is the intended reactive outcome. However, the role of NHS esters as activated carboxyls can also drive the formation of serine-, tyrosine-, and threonine-derived esters (O-derivatives). These O-derivative peptides are typically classed as overlabeled and are disregarded for quantitation, leading to loss of information and hence potential sensitivity. Their presence also unnecessarily increases sample complexity, which reduces the overall identification rates. One common approach for removing these unwanted labeling events has involved treatment with hydroxylamine. We show here that this approach is not efficient and can still leave substantial levels of unwanted overlabeled peptides. Through systematic study of nucleophilic aminolysis reagents and reaction conditions, we have now developed a robust method to efficiently remove overlabeled peptides. The new method reduces the proportion of overlabeled peptides in the sample to less than 1% without affecting the labeling rate or introducing other modifications, leading to superior identification rates and quantitation precision.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100948"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004380/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metal ion-enhanced ZIC-cHILIC StageTip for N-Glycoproteomic and Phosphoproteomic Profiling in EGFR-mutated Lung Cancer Cells.","authors":"Yi-Ju Chen, Yan-Lin Chen, Kun-Hao Chang, Hsiang-Chun Cheng, Chiao-Chun Chang, Yu-Ju Chen","doi":"10.1016/j.mcpro.2025.100957","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100957","url":null,"abstract":"<p><p>Surface glycosylation and intracellular phosphorylation regulates the cell-cell communication and signaling cascades. Due to complex glycosylation and dynamic phosphorylation, exploring their interplay remains technically challenging. In this study, we reported a tandem ZIC-cHILIC StageTip strategy for streamlined and simultaneous (sialo)glycoproteomic and phosphoproteomic profiling. We first demonstrated that Fe ions expand the utility of ZIC-cHILIC strategy to phosphoproteomic analysis with greatly enhanced >4-fold coverage and high specificity for mono-phosphopeptides (95%). The Fe-ZIC-cHILIC tandem tips, leveraging stepwise fractionation, enable large-scale coverage of 10,536 glycopeptides, including highly confident 4,285 sialoglycopeptpides, and 11,329 phosphopeptides in a single cell type. To study the mechanism underlying the tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC), application of the strategy to 4 NSCLC cells harboring different EGFR mutations reveals significantly differential 1,559 glycopeptides and 1,949 phosphopeptides either in EGFR mutation or TKI resistant cells. Without protein immunoprecipitation, the approach identified FDA-approved drug targets, such as EGFR, ERBB2, MET, and integrin family members. Most prominent alterations were observed in EGFR (auto-phosphorylation Y1197 and 10 bi- and triantennary fucosyl-sialo glycans at N603), downstream PI3K-Akt pathway (ERBB2-T1240, MET-S990/T992, AKT-S124/S126) and integrin family (sialo-fucosyl glycans), suggesting site-specific alteration between N-glycosylation and phosphorylation interplay in the TKI resistant L858R-T790M mutant NSCLC cells. The glycoproteomic and phosphoproteomic landscape may help to unravel the complex modification alterations underlying the resistant mechanism, offering insights for improving therapeutic strategies and patient outcomes.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100957"},"PeriodicalIF":6.1,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143743185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Single-Step Protein Extraction for Lung Extracellular Matrix Proteomics Enabled by the Photocleavable Surfactant Azo and timsTOF Pro.","authors":"Anna G Towler, Andrew J Perciaccante, Timothy J Aballo, Yanlong Zhu, Fei Wang, Sarah Lloyd, Kuniko Kadoya, Yupeng He, Yu Tian, Ying Ge","doi":"10.1016/j.mcpro.2025.100950","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100950","url":null,"abstract":"<p><p>The extracellular matrix (ECM) is a dynamic, complex network of proteins, collectively known as the 'matrisome', which not only provides essential structural support to cells and tissues but also regulates critical cellular processes. Dysregulation of the ECM is implicated in many diseases, underscoring the need to characterize the matrisome to better understand disease mechanisms. We have previously developed a dual-step protocol enabled by the photocleavable surfactant Azo for the extraction of ECM proteins from tissue using pH-neutral decellularization followed by solubilization by Azo. While effective for characterization of the ECM proteins, such a dual-step protocol requires two extracts per sample, limiting the throughput and complicating the comparison of protein quantitation across different extraction conditions. Here, we develop a single-step Azo-enabled protein extraction for the solubilization of ECM proteins from lung tissue to improve the throughput for studies with large sample sizes. Using this method, we identified 324 ECM proteins, including 137 core ECM and 187 ECM associated proteins. Core ECM proteins including elastin, fibronectin, and fibrillar collagens were reproducibly identified and quantified. We observed a 94.6% overlap in the ECM proteins identified between the single-step and dual-step Azo extracts, indicating the single-step Azo extraction achieves ECM protein coverage comparable to the dual-step extraction. Overall, we have demonstrated that this single-step Azo extraction is not only highly efficient but also comprehensive for ECM protein identification and quantification, making it a powerful method for ECM proteomics, especially for studies with large sample size.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100950"},"PeriodicalIF":6.1,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive proteomics metadata and integrative web portals facilitate sharing and integration of LINCS multiomics data.","authors":"Dušica Vidović, Behrouz Shamsaei, Stephan C Schürer, Phillip Kogan, Szymon Chojnacki, Michal Kouril, Mario Medvedovic, Wen Niu, Evren U Azeloglu, Marc R Birtwistle, Yibang Chen, Tong Chen, Jens Hansen, Bin Hu, Ravi Iyengar, Gomathi Jayaraman, Hong Li, Tong Liu, Eric A Sobie, Yuguang Xiong, Matthew J Berberich, Gary Bradshaw, Mirra Chung, Robert A Everley, Ben Gaudio, Marc Hafner, Marian Kalocsay, Caitlin E Mills, Maulik K Nariya, Peter K Sorger, Kartik Subramanian, Chiara Victor, Maria Banuelos, Victoria Dardov, Ronald Holewinski, Danica-Mae Manalo, Berhan Mandefro, Andrea D Matlock, Loren Ornelas, Dhruv Sareen, Clive N Svendsen, Vineet Vaibhav, Jennifer E Van Eyk, Vidya Venkatraman, Steve Finkbiener, Ernest Fraenkel, Jeffrey Rothstein, Leslie Thompson, Jacob Asiedu, Steven A Carr, Karen E Christianson, Desiree Davison, Deborah O Dele-Oni, Katherine C DeRuff, Shawn B Egri, Alvaro Sebastian Vaca Jacome, Jacob D Jaffe, Daniel Lam, Lev Litichevskiy, Xiaodong Lu, James Mullahoo, Adam Officer, Malvina Papanastasiou, Ryan Peckner, Caidin Toder, Joel Blanchard, Michael Bula, Tak Ko, Li-Huei Tsai, Jennie Z Young, Vagisha Sharma, Ajay Pillai, Jarek Meller, Michael J MacCoss","doi":"10.1016/j.mcpro.2025.100947","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100947","url":null,"abstract":"<p><p>The Library of Integrated Network-based Cellular Signatures (LINCS), an NIH Common Fund program, has cataloged and analyzed cellular function and molecular activity profiles in response to >80,000 perturbing agents that are potentially disruptive to cells. Because of the importance of proteins and their modifications to the response of specific cellular perturbations, four of the six LINCS centers have included significant proteomics efforts in the characterization of the resulting phenotype. This manuscript aims to describe this effort and the data harmonization and integration of the LINCS proteomics data discussed in recent LINCS papers.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100947"},"PeriodicalIF":6.1,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deep Learning Enhances Precision of Citrullination Identification in Human and Plant Tissue Proteomes.","authors":"Wassim Gabriel, Rebecca Meelker González, Sophia Laposchan, Erik Riedel, Gönül Dündar, Brigitte Poppenberger, Mathias Wilhelm, Chien-Yun Lee","doi":"10.1016/j.mcpro.2025.100924","DOIUrl":"10.1016/j.mcpro.2025.100924","url":null,"abstract":"<p><p>Citrullination is a critical yet understudied post-translational modification (PTM) implicated in various biological processes. Exploring its role in health and disease requires a comprehensive understanding of the prevalence of this PTM at a proteome-wide scale. Although mass spectrometry has enabled the identification of citrullination sites in complex biological samples, it faces significant challenges, including limited enrichment tools and a high rate of false positives due to the identical mass with deamidation (+0.9840 Da) and errors in monoisotopic ion selection. These issues often necessitate manual spectrum inspection, reducing throughput in large-scale studies. In this work, we present a novel data analysis pipeline that incorporates the deep learning model Prosit-Cit into the MS database search workflow to improve both the sensitivity and the precision of citrullination site identification. Prosit-Cit, an extension of the existing Prosit model, has been trained on ∼53,000 spectra from ∼2500 synthetic citrullinated peptides and provides precise predictions for chromatographic retention time and fragment ion intensities of both citrullinated and deamidated peptides. This enhances the accuracy of identification and reduces false positives. Our pipeline demonstrated high precision on the evaluation dataset, recovering the majority of known citrullination sites in human tissue proteomes and improving sensitivity by identifying up to 14 times more citrullinated sites. Sequence motif analysis revealed consistency with previously reported findings, validating the reliability of our approach. Furthermore, extending the pipeline to a tissue proteome dataset of the model plant Arabidopsis thaliana enabled the identification of ∼200 citrullination sites across 169 proteins from 30 tissues, representing the first large-scale citrullination mapping in plants. This pipeline can be seamlessly applied to existing proteomics datasets, offering a robust tool for advancing biological discoveries and deepening our understanding of protein citrullination across species.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100924"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11925583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Primer on Proteomic Characterization of Intercellular Communication in a Virus Microenvironment.","authors":"James C Kostas, Colter S Brainard, Ileana M Cristea","doi":"10.1016/j.mcpro.2025.100913","DOIUrl":"10.1016/j.mcpro.2025.100913","url":null,"abstract":"<p><p>Intercellular communication is fundamental to multicellular life and a core determinant of outcomes during viral infection, where the common goals of virus and host for persistence and replication are generally at odds. Hosts rely on encoded innate and adaptive immune responses to detect and clear viral pathogens, while viruses can exploit or disrupt these pathways and other intercellular communication processes to enhance their spread and promote pathogenesis. While virus-induced signaling can result in systemic changes to the host, striking alterations are observed within the cellular microenvironment directly surrounding a site of infection, termed the virus microenvironment (VME). Mechanisms employed by viruses to condition their VMEs are emerging and are critical for understanding the biology and pathologies of viral infections. Recent advances in experimental approaches, including proteomic methods, have enabled study of the VME in unprecedented detail. In this review article, we provide a primer on proteomic approaches used to study how viral infections alter intercellular communication, highlighting the ways in which these approaches have been implemented and the exciting biology they have uncovered. First, we consider the different molecules secreted by an infected cell, including proteins, either soluble or contained within extracellular vesicles, and metabolites. We further discuss the modalities of interactions facilitated by alteration at the cell surface of infected cells, including immunopeptide presentation and interactions with the extracellular matrix. Finally, we review spatial profiling approaches that have allowed distinguishing how specific subpopulations of cells within a VME respond to infection and alter their protein composition, discussing valuable insights these methods have offered.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100913"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11889360/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143040241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation of Proteins on Chromatin Reveals Signaling Pathway-Dependent Alterations in the DNA-Bound Proteome.","authors":"Huiyu Wang, Azmal Ali Syed, Jeroen Krijgsveld, Gianluca Sigismondo","doi":"10.1016/j.mcpro.2025.100908","DOIUrl":"10.1016/j.mcpro.2025.100908","url":null,"abstract":"<p><p>Signaling pathways often convergence on transcription factors and other DNA-binding proteins that regulate chromatin structure and gene expression, thereby governing a broad range of essential cellular functions. However, the repertoire of DNA-binding proteins is incompletely understood even for the best-characterized pathways. Here, we optimized a strategy for the isolation of Proteins on Chromatin (iPOC) exploiting tagged nucleoside analogs to label the DNA and capture associated proteins, thus enabling the comprehensive, sensitive, and unbiased characterization of the DNA-bound proteome. We then applied iPOC to investigate chromatome changes upon perturbation of the cancer-relevant PI3K-AKT-mTOR pathway. Our results show distinct dynamics of the DNA-bound proteome upon selective inhibition of PI3K, AKT, or mTOR, and we provide evidence how this signaling cascade regulates the DNA-bound status of SUZ12, thereby modulating H3K27me3 levels. Collectively, iPOC is a powerful approach to study the composition of the DNA-bound proteome operating downstream of signaling cascades, thereby both expanding our knowledge of the mechanism of action of the pathway and unveiling novel chromatin modulators that can potentially be targeted pharmacologically.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100908"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11889358/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143023049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative Analysis of the Total Proteome in Nonalcoholic Steatohepatitis: Identification of Potential Biomarkers.","authors":"Eda Ates, Hien Thi My Ong, Seung-Min Yu, Ji-Hoon Kim, Min-Jung Kang","doi":"10.1016/j.mcpro.2025.100921","DOIUrl":"10.1016/j.mcpro.2025.100921","url":null,"abstract":"<p><p>Nonalcoholic fatty liver disease is a hepatic condition characterized by excessive fat accumulation in the liver with advanced stage nonalcoholic steatohepatitis (NASH), potentially leading to liver fibrosis, cirrhosis, and cancer. Currently, the identification and classification of NASH require invasive liver biopsy, which has certain limitations. Mass spectrometry-based proteomics can detect crucial proteins and pathways implicated in NASH development and progression. We collected the liver and serum samples from choline-deficient, L-amino acid-defined high-fat diet fed NASH C57BL/6J mice and human serum samples to examine proteomic alterations and identify early biomarkers for NASH diagnosis. In-depth targeted multiple reaction monitoring scanning and immunoblotting assays were used to verify the biomarker candidates from mouse liver and serum samples, and enzyme-linked immunosorbent assay (ELISA) was employed to analyze human serum samples. The multiple reaction monitoring analysis of NASH liver revealed 50 proteins with altered expression (21 upregulated and 29 downregulated) that are involved in biological processes such as detoxification, fibrosis, inflammation, and fatty acid metabolism. Ingenuity pathway analysis identified impaired protein synthesis, cellular stress and defense, cellular processes and communication, and metabolism in NASH mouse liver. Immunoblotting analysis confirmed that the expression of proteins associated with fatty acid metabolism (Aldo B and Fasn) and urea cycle (Arg1, Cps1, and Otc) was altered in the mouse liver and serum. Further analysis on human serum samples using ELISA confirmed the increased expression of multiple proteins, including Aldo B, Asl, and Lgals3, demonstrating values of 0.917, 0.979, and 0.965 of area under the curve in NASH diagnosis. These findings offer valuable insights into the molecular mechanisms of NASH and possible diagnostic biomarkers for early detection.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100921"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11910689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143080366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PathwayPilot: A User-Friendly Tool for Visualizing and Navigating Metabolic Pathways.","authors":"Tibo Vande Moortele, Pieter Verschaffelt, Qingyao Huang, Nadezhda T Doncheva, Tanja Holstein, Caroline Jachmann, Peter Dawyndt, Lennart Martens, Bart Mesuere, Tim Van Den Bossche","doi":"10.1016/j.mcpro.2025.100918","DOIUrl":"10.1016/j.mcpro.2025.100918","url":null,"abstract":"<p><p>Metaproteomics, the study of collective proteomes in environmental communities, plays a crucial role in understanding microbial functionalities affecting ecosystems and human health. Pathway analysis offers structured insights into the biochemical processes within these communities. However, no existing tool effectively combines pathway analysis with peptide- or protein-level data. We here introduce PathwayPilot, a web-based application designed to improve metaproteomic data analysis by integrating pathway analysis with peptide- and protein-level data, filling a critical gap in current metaproteomics bioinformatics tools. By allowing users to compare functional annotations across different samples or multiple organisms within a sample, PathwayPilot provides valuable insights into microbial functions. In the re-analysis of a study examining the effects of caloric restriction on gut microbiota, the tool successfully identified shifts in enzyme expressions linked to short-chain fatty acid biosynthesis, aligning with its original findings. PathwayPilot's user-friendly interface and robust capabilities make it a significant advancement in metaproteomics, with the potential for widespread application in microbial ecology and health sciences. All code is open source under the Apache2 license and is available at https://pathwaypilot.ugent.be.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100918"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11903815/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143066265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive Immunoglobulin G, A, and M Glycopeptide Profiling for Large-Scale Biomedical Research.","authors":"Bianca D M van Tol, Anna M Wasynczuk, Steinar Gijze, Oleg A Mayboroda, Jan Nouta, Radboud J E M Dolhain, Manfred Wuhrer, David Falck","doi":"10.1016/j.mcpro.2025.100928","DOIUrl":"10.1016/j.mcpro.2025.100928","url":null,"abstract":"<p><p>Glycosylation of immunoglobulin G (IgG) is recognized as a key modulator of cellular effector functions. At the same time, an increasing body of evidence underlines the importance of other antibody isotypes, especially IgA and IgM, in pathophysiological conditions. Therefore, methods to efficiently study the complex interplay between isotypes, subclasses, and glycosylation of antibodies during acute and chronic states of inflammation are needed. As a solution, we present an integrated and comprehensive method combining simultaneous affinity enrichment of IgG, IgA, and IgM with a single measurement, glycopeptide-centered LC-MS analysis of all isotypes which provides protein-specific (isotype and subclass), and site-specific N- and O-glycosylation quantitation. A two-protease approach provided individual peptides for each glycosylation site, allowing unambiguous compositional assignment and relative quantitation of glycoforms on the MS¹ level as well as structural confirmation and partial isomer assignment on the MS/MS level. We demonstrate that our methodology can be efficiently applied to large clinical studies revealing differences in antibody glycosylation in women during and after pregnancy, as well as between healthy donors and patients with rheumatoid arthritis. In addition, this showcased the advantages of our method in comprehensiveness and resolution of isotypes, subclasses, and glycosylation sites as well as its precision and robustness.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100928"},"PeriodicalIF":6.1,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11953977/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143472691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}