Molecular & Cellular Proteomics最新文献

{"title":"PEPSeek-Mediated Identification of Novel Epitopes From Viral and Bacterial Pathogens and the Impact on Host Cell Immunopeptidomes.","authors":"John A Cormican, Lobna Medfai, Magdalena Wawrzyniuk, Martin Pašen, Hassnae Afrache, Constance Fourny, Sahil Khan, Pascal Gneiße, Wai Tuck Soh, Arianna Timelli, Emanuele Nolfi, Yvonne Pannekoek, Andrew Cope, Henning Urlaub, Alice J A M Sijts, Michele Mishto, Juliane Liepe","doi":"10.1016/j.mcpro.2025.100937","DOIUrl":"10.1016/j.mcpro.2025.100937","url":null,"abstract":"<p><p>Here, we develop PEPSeek, a web-server-based software to allow higher performance in the identification of pathogen-derived epitope candidates detected via mass spectrometry in MHC class I immunopeptidomes. We apply it to human and mouse cell lines infected with SARS-CoV-2, Listeria monocytogenes, or Chlamydia trachomatis, thereby identifying a large number of novel antigens and epitopes that we prove to be recognized by CD8⁺ T cells. In infected cells, we identified antigenic peptide features that suggested how the processing and presentation of pathogenic antigens differ between pathogens. The quantitative tools of PEPSeek also helped to define how C. trachomatis infection cycle could impact the antigenic landscape of the host human cell system, likely reflecting metabolic changes that occurred in the infected cells.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100937"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12002930/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567530","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":"Analysis of Limited Proteolysis-Coupled Mass Spectrometry Data.","authors":"Luise Nagel, Jan Grossbach, Valentina Cappelletti, Christian Dörig, Paola Picotti, Andreas Beyer","doi":"10.1016/j.mcpro.2025.100934","DOIUrl":"10.1016/j.mcpro.2025.100934","url":null,"abstract":"<p><p>Limited proteolysis combined with mass spectrometry (LiP-MS) facilitates probing structural changes on a proteome-wide scale. This method leverages differences in the proteinase K accessibility of native protein structures to concurrently assess structural alterations for thousands of proteins in situ. Distinguishing different contributions to the LiP-MS signal, such as changes in protein abundance or chemical modifications, from structural protein alterations remains challenging. Here, we present the first comprehensive computational pipeline to infer structural alterations for LiP-MS data using a two-step approach. 1) We remove unwanted variations from the LiP signal that are not caused by protein structural effects and 2) infer the effects of variables of interest on the remaining signal. Using LiP-MS data from three species, we demonstrate that this approach outperforms previously employed approaches. Our framework provides a uniquely powerful approach for deconvolving LiP-MS signals and separating protein structural changes from changes in protein abundance, posttranslational modifications, and alternative splicing. Our approach may also be applied to analyze other types of peptide-centric structural proteomics data, such as FPOP or molecular painting data.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100934"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12036054/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586240","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":"Integrated Multiomics Reveals Alterations in Paucimannose and Complex Type N-Glycans in Cardiac Tissue of Patients with COVID-19.","authors":"Sabarinath Peruvemba Subramanian, Melinda Wojtkiewicz, Fang Yu, Chase Castro, Erin N Schuette, Jocelyn Rodriguez-Paar, Jared Churko, Pranav Renavikar, Daniel Anderson, Claudius Mahr, Rebekah L Gundry","doi":"10.1016/j.mcpro.2025.100929","DOIUrl":"10.1016/j.mcpro.2025.100929","url":null,"abstract":"<p><p>Coronavirus infectious disease of 2019 (COVID-19) can lead to cardiac complications, yet the molecular mechanisms driving these effects remain unclear. Protein glycosylation is crucial for viral replication, immune response, and organ function and has been found to change in the lungs and liver of patients with COVID-19. However, how COVID-19 impacts cardiac protein glycosylation has not been defined. Our study combined single nuclei transcriptomics, mass spectrometry (MS)-based glycomics, and lectin-based tissue imaging to investigate alterations in N-glycosylation in the human heart post-COVID-19. We identified significant expression differences in glycogenes involved in N-glycan biosynthesis and MS analysis revealed a reduction in high mannose and isomers of paucimannose structures post-infection, with changes in paucimannose directly correlating with COVID-19 independent of comorbidities. Our observations suggest that COVID-19 primes cardiac tissues to alter the glycome at all levels, namely, metabolism, nucleotide sugar transport, and glycosyltransferase activity. Given the role of N-glycosylation in cardiac function, this study provides a basis for understanding the molecular events leading to cardiac damage post-COVID-19 and informing future therapeutic strategies to treat cardiac complications resulting from coronavirus infections.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100929"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12131855/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483492","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":"Proteomics Analysis of Porcine Endometrial Cell-Derived Extracellular Vesicles Involved in Embryo Attachment.","authors":"Seonggyu Bang, Ahmad Yar Qamar, Sang-Yeop Lee, Ayeong Han, Heejae Kang, Bereket Molla Tanga, Sung Ho Yun, Hye Sun Park, Seung Il Kim, Won Gi Yoo, Islam M Saadeldin, Sanghoon Lee, Jongki Cho","doi":"10.1016/j.mcpro.2025.100942","DOIUrl":"10.1016/j.mcpro.2025.100942","url":null,"abstract":"<p><p>Maternal-embryo interactions play a critical role in early mammalian development, with extracellular vesicles (EVs) playing a key role in intercellular communication. Recent studies have focused on the mechanisms by which maternal-derived factors, such as RNA, proteins, and metabolites influence gap junctions, EVs, and direct cell-to-cell interactions, contributing to embryonic development. In this study, using a proteomics approach, we investigated the impact of EVs secreted from porcine endometrial cells (pEECs) and their protein cargoes on embryonic development. We characterized EVs isolated from pEECs (pEEC-EVs) during the diestrus stage using a nanoparticle tracking analysis and cryo-transmission electron microscopy. Furthermore, the effects of pEEC-EVs with or without hormone treatment on the in vitro attachment of hatched blastocysts were evaluated. The attachment rate of porcine embryos was significantly higher for pEEC-EVs in the hormone treatment group than the control group (23.0 ± 1.7% versus 36.9 ± 1.9% for control and pEEC-EVs, respectively). Furthermore, hormone treatment altered the expression of proteins involved in cellular organization, protein transport, and immunity. Proteomic analysis revealed distinct biological processes between groups: control EVs supported cytoskeletal organization and adhesion, while hormone-treated EVs were enriched in protein transport, immune regulation, and stress response pathways. Key signaling pathways, including VEGFA-VEGFR2, focal adhesion, and TGF-β, were modulated, influencing implantation and embryogenesis. EVs play a crucial role in maternal-embryo interactions, optimizing implantation conditions and supporting embryo-derived stem cell establishment. These findings enhance our understanding of EV-mediated communication and suggest potential applications for improving reproductive health and assisted reproductive technologies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100942"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004383/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625465","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":"diaPASEF Analysis for HLA-I Peptides Enables Quantification of Common Cancer Neoantigens.","authors":"Denys Oliinyk, Hem R Gurung, Zhenru Zhou, Kristin Leskoske, Christopher M Rose, Susan Klaeger","doi":"10.1016/j.mcpro.2025.100938","DOIUrl":"10.1016/j.mcpro.2025.100938","url":null,"abstract":"<p><p>Human leukocyte antigen class I (HLA-I) molecules present short peptide sequences from endogenous or foreign proteins to cytotoxic T cells. The low abundance of HLA-I peptides poses significant technical challenges for their identification and accurate quantification. While mass spectrometry is currently a method of choice for direct system-wide identification of cellular immunopeptidomes, there is still a need for enhanced sensitivity in detecting and quantifying tumor-specific epitopes. As gas phase separation in data-dependent MS data acquisition increased HLA-I peptide detection by up to 50%, here, we aimed to evaluate the performance of data-independent acquisition (DIA) in combination with parallel accumulation serial fragmentation ion mobility (diaPASEF) for high-sensitivity identification of HLA presented peptides. Our streamlined diaPASEF workflow enabled identification of 11,412 unique peptides from 12.5 million A375 cells and 3426 8-11mers from as low as 500,000 cells with high reproducibility. By taking advantage of HLA binder-specific in silico predicted spectral libraries, we were able to further increase the number of identified HLA-I peptides. We applied SILAC-DIA to a mixture of labeled HLA-I peptides, calculated heavy-to-light ratios for 7742 peptides across five conditions and demonstrated that diaPASEF achieves high quantitative accuracy up to 5-fold dilution. Finally, we identified and quantified shared neoantigens in a monoallelic C1R cell line model. By spiking in heavy synthetic peptides, we verified the identification of the peptide sequences and calculated relative abundances for 13 neoantigens. Taken together, diaPASEF analysis workflows for HLA-I peptides can increase the peptidome coverage for lower sample amounts. The sensitivity and quantitative precision provided by DIA can enable the detection and quantification of less abundant peptide species such as neoantigens across samples from the same background.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100938"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12003003/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143567529","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":"Proteomics and Machine Learning-Based Approach to Decipher Subcellular Proteome of Mouse Heart.","authors":"Haoyun Fang, Alin Rai, Seyed Sadegh Eslami, Kevin Huynh, Hsiao-Chi Liao, Agus Salim, David W Greening","doi":"10.1016/j.mcpro.2025.100952","DOIUrl":"10.1016/j.mcpro.2025.100952","url":null,"abstract":"<p><p>Protein compartmentalization to distinctive subcellular niches is critical for cardiac function and homeostasis. Here, we employed a rapid and robust workflow based on differential centrifugal-based fractionation with mass spectrometry-based proteomics and bioinformatic analyses for systemic mapping of the subcellular proteome of mouse heart. Using supervised machine learning of 450 hallmark protein markers from 16 subcellular niches, we further refined the subcellular information of 2083 proteins with high confidence. Our data validation focused on specific subcellular niches such as mitochondria, cell surface, cardiac dyad, myofibril, and nuclear, unfolding dominant subcellular localization of proteins in their native environment of mouse heart. We further provide targeted nuclear enrichment and co-immunoprecipitation-based proteomic validation from the heart of nuclear-localizing protein networks. This study provides novel insights into the molecular landscape of different subcellular niches of the heart and serves as a draft map for heart subcellular proteome.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100952"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12019842/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670496","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":"Embryo-Induced Changes in the Protein Profile of Bovine Oviductal Extracellular Vesicles.","authors":"Rosane Mazzarella, José María Sánchez, Beatriz Fernandez-Fuertes, Sandra Guisado Egido, Michael McDonald, Alberto Álvarez-Barrientos, Esperanza González, Juan Manuel Falcón-Pérez, Mikel Azkargorta, Félix Elortza, Maria Encina González, Pat Lonergan, Dimitrios Rizos","doi":"10.1016/j.mcpro.2025.100935","DOIUrl":"10.1016/j.mcpro.2025.100935","url":null,"abstract":"&lt;p&gt;&lt;p&gt;The study of early maternal-embryonic cross-talk remains one of the most challenging topics in reproductive biology. Understanding the physiological mechanisms involved in the interactions between the maternal reproductive tract and the developing embryo is essential for enhancing bovine reproductive efficiency. This complex communication starts within the oviduct, where the modulation of biological processes important for ensuring embryo quality is partially facilitated through extracellular vesicles (EVs). Utilizing a combination of in vivo and in vitro models this study had three main objectives: 1) to examine the protein cargo of EVs isolated from the oviductal fluid (OF) of cyclic and pregnant heifers to understand their role in maternal-embryonic communication in vivo; 2) to characterize the protein profile of EVs in conditioned medium (CM) resulting from the culture of oviductal explants alone (Exp) or in the presence of 8- to 16-cell stage embryos (Exp + Emb); and 3) to compare the protein cargo of EVs from Exp with EVs from cyclic heifers and EVs from Exp + Emb with EVs from pregnant heifers. Proteins were considered \"identified\" if detected in at least three out of five replicates and considered \"exclusive\" if detected in at least three out of five replicates within one group but absent in all samples of other groups. We identified 659 and 1476 proteins in the OF-EVs of cyclic and pregnant heifers, respectively. Among these, 644 proteins were identified in OF-EVs from both cyclic and pregnant heifers, and 40 proteins were exclusive to OF-EVs from the pregnant group. Within the 644 proteins identified in both groups, 31 were identified as differently abundant proteins (DAPs). In pregnant heifers, DAPs were mainly related to genome activation, DNA repair, embryonic cell differentiation, migration, and immune tolerance. In vitro, we identified 841 proteins in the CM-EVs from Exp alone, 613 from Exp + Emb, and 111 in the CM-EVs from Emb alone. In the qualitative analysis between the three in vitro groups, 81 proteins were identified in all groups, 452 were common to Exp and Exp + Emb, 17 were common to Exp and Emb, 5 were common to Exp + Emb and Emb, 4 were unique to Exp, 6 were unique to Exp + Emb, and none were unique to Emb. Proteins identified when there is an interaction between the oviduct and the embryo in vitro, corresponding to the Exp + Emb group, were associated with immune tolerance, structural activity, binding, and cytoskeletal regulation. In vivo and in vitro EVs exhibit distinct qualitative and quantitative protein contents, both when comparing EVs produced in the absence of an embryo (Cyclic and Exp) and those that have undergone embryo-oviduct interaction (Pregnant and Exp + Emb). The observed changes in the protein cargo of EVs due to maternal-embryonic communication in vivo and in vitro suggest that the interaction between the embryo and the maternal milieu initiates within the oviduct and is potentially facilitate","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100935"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11994978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537410","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":"Benchmarking of Quantitative Proteomics Workflows for Limited Proteolysis Mass Spectrometry.","authors":"Tomas Koudelka, Claudio Bassot, Ilaria Piazza","doi":"10.1016/j.mcpro.2025.100945","DOIUrl":"10.1016/j.mcpro.2025.100945","url":null,"abstract":"<p><p>Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for detecting protein structural changes and drug-protein interactions on a proteome-wide scale. However, there is no consensus on the best quantitative proteomics workflow for analyzing LiP-MS data. In this study, we comprehensively benchmarked two major quantification approaches-data-independent acquisition (DIA) and tandem mass tag (TMT) isobaric labeling-in combination with LiP-MS, using a drug-target deconvolution assay as a model system. Our results show that while TMT labeling enabled the quantification of more peptides and proteins with lower coefficients of variation, DIA-MS exhibited greater accuracy in identifying true drug targets and stronger dose-response correlation in peptides of protein targets. Additionally, we evaluated the performance of freely available (FragPipe) versus commercial (Spectronaut) software tools for DIA-MS analysis, revealing that the choice between precision (FragPipe) and sensitivity (Spectronaut) largely depends on the specific experimental context. Our findings underscore the importance of selecting the appropriate LiP-MS quantification strategy based on the study objectives. This work provides valuable guidelines for researchers in structural proteomics and drug discovery, and highlights how advancements in mass spectrometry instrumentation, such as the Astral mass spectrometer, may further improve sensitivity and protein sequence coverage, potentially reducing the need for TMT labeling.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100945"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12022698/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634287","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":"Proteomic Analysis of Aqueous Humor Identified Clinically Relevant Molecular Targets for Neovascular Complications in Diabetic Retinopathy.","authors":"Jae Won Oh, Seong Joon Ahn, Jae Hun Jung, Tae Wan Kim, Kwang Pyo Kim","doi":"10.1016/j.mcpro.2025.100953","DOIUrl":"10.1016/j.mcpro.2025.100953","url":null,"abstract":"<p><p>Diabetic retinopathy (DR) is a leading cause of blindness in adults under 40 in the developed world, with a significant proportion progressing to vision-threatening stages such as proliferative diabetic retinopathy (PDR) and neovascular glaucoma (NVG). This study aims to explore the molecular mechanisms underlying the progression from nonproliferative DR to PDR and NVG, focusing on identifying potential biomarkers and therapeutic targets. Utilizing discovery-based proteomics, specifically label-free quantification and tandem mass tag, we analyzed aqueous humor (AH) proteins obtained during cataract surgery or anterior chamber paracentesis from patients with nonproliferative DR, PDR, and NVG. Validation of marker candidates for each disease state was conducted using triple quadrupole-MS for targeted protein quantification. Our proteomic analysis identified 2255 proteins, and gene ontology analysis and functional annotation highlighted key biological processes implicated in DR, such as lens development, immune responses, and lipid metabolism. Validation of potential biomarkers identified 20 proteins with significant concentration changes, including several candidates with diagnostic utility based on ROC curve analysis. Further investigation into clinical relevance revealed that crystallin gamma-S is strongly associated with cataract severity, highlighting its role as a potential marker for ocular complications in DR. Importantly, we identified that the pathological factors driving DR progression have a much greater impact than age, a previously known variable, in shaping the proteomic landscape of AH. Additionally, proteins associated with macular degeneration (CA1, CA2, and HBA1) were uncovered, providing new insights into overlapping mechanisms between DR and other retinal diseases. Finally, proteins linked to panretinal photocoagulation treatment, including APOB and CST6, were identified, suggesting their involvement in the therapeutic response and post-treatment adaptation. These findings underscore the potential of AH proteomics in uncovering predictive biomarkers and elucidating the molecular pathogenesis of DR and its complications.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100953"},"PeriodicalIF":6.1,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12131854/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674394","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":"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}