Molecular & Cellular Proteomics最新文献

{"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":"https://doi.org/10.1016/j.mcpro.2025.100952","url":null,"abstract":"<p><p>Protein compartmentalisation 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 (MS)-based proteomics and bioinformatic analyses for systemic mapping of the subcellular proteome of mouse heart. Using supervised machine learning (ML) 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 localisation 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-localising 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":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Deleterious knock-outs in the HLA class I antigen processing and presentation machinery induce distinct changes in the immunopeptidome.","authors":"Ilja E Shapiro, Clélia Maschke, Justine Michaux, HuiSong Pak, Laura Wessling, Tamara Verkerk, Robbert Spaapen, Michal Bassani-Sternberg","doi":"10.1016/j.mcpro.2025.100951","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100951","url":null,"abstract":"<p><p>The human leukocyte antigen (HLA) processing and presentation machinery (APPM) is altered in various diseases and in response to drug treatments. Defects in the machinery may change presentation levels or alter the repertoire of presented peptides, globally or in an HLA allele restricted manner, with direct implications for adaptive immunity. In this study, we investigated the immunopeptidome landscape across a panel of isogenic HAP1 cell line clones each with a knock-out of a single gene encoding a key protein in the APPM, including B2M, TAP1, TAP2, TAPBP, IRF2, PDIA3, ERAP1, GANAB, SPPL3, CANX, and CALR. We applied immunopeptidomic and proteomic to assess the successful gene knock-outs on the protein level, to understand how these proteins participate in antigen presentation, and to contextualize protein expression and antigen presentation. We validated the absence of the knocked-out proteins in the respective samples and found that knocking-out an APPM component leads to the loss of peptide subsets that are normally presented on the control wild type cells. We assessed the immunopeptidomes qualitatively and quantitatively, considering factors like peptide diversity, peptide length distribution, and binding affinity to the endogenously expressed HLA alleles in HAP1 cells. We demonstrated a prominent HLA allele-specific alterations in several knock-out conditions. The absence of CALR, CANX, and TAP1 led to significant changes in HLA allele-specific presentation levels. Overall, this work represents the first systematic analysis of how the absence of individual APPM components, knocked out in a single cell line under controlled conditions, affects the peptidome. This approach could facilitate the creation of predictive tools capable of prioritizing HLA-bound peptides likely to be presented when presentation defects occur, such as in cancer and viral infections.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100951"},"PeriodicalIF":6.1,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670495","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":"Single-Cell Proteomic Characterization of Drug-Resistant Prostate Cancer Cells Reveals Molecular Signatures Associated with Morphological Changes.","authors":"Jongmin Woo, Michael Loycano, Md Amanullah, Jiang Qian, Sarah R Amend, Kenneth J Pienta, Hui Zhang","doi":"10.1016/j.mcpro.2025.100949","DOIUrl":"10.1016/j.mcpro.2025.100949","url":null,"abstract":"<p><p>This study delves into the proteomic intricacies of drug-resistant cells (DRCs) within prostate cancer, which are known for their pivotal roles in therapeutic resistance, relapse, and metastasis. Utilizing single-cell proteomics (SCP) with an optimized high-throughput data-independent acquisition (DIA) approach with the throughput of 60 sample per day, we characterized the proteomic landscape of DRCs in comparison to parental PC3 cells. This DIA method allowed for robust and reproducible protein quantification at the single-cell level, enabling the identification and quantification of over 1300 proteins per cell on average. Distinct proteomic sub-clusters within the DRC population were identified, closely linked to variations in cell size. The study uncovered novel protein signatures, including the regulation of proteins critical for cell adhesion and metabolic processes, as well as the upregulation of surface proteins and transcription factors pivotal for cancer progression. Furthermore, by conducting single-cell RNA-seq (scRNA-seq) analysis, we identified six upregulated and 10 downregulated genes consistently altered in drug-treated cells across both SCP and scRNA-seq platforms. These findings underscore the heterogeneity of DRCs and their unique molecular signatures, providing valuable insights into their biological behavior and potential therapeutic targets.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100949"},"PeriodicalIF":6.1,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143639750","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":"Metabolic Reprogramming into a Glycolysis Phenotype Induced by Extracellular Vesicles Derived from Prostate Cancer Cells.","authors":"Yoon-Jin Lee, Chul Won Seo, Shinwon Chae, Chang Yeol Lee, Sang Soo Kim, Yoon-Hee Shin, Hyun-Mee Park, Yong Song Gho, Seongho Ryu, Sang-Han Lee, Dongsic Choi","doi":"10.1016/j.mcpro.2025.100944","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100944","url":null,"abstract":"<p><p>Most cancer cells adopt a less efficient metabolic process of aerobic glycolysis with high level of glucose uptake followed by lactic acid production, known as the Warburg effect. This phenotypic transition enables cancer cells to achieve increased cellular survival and proliferation in a harsh low-oxygen tumor microenvironment. Also, the resulting acidic microenvironment causes inactivation of the immune system such as T-cell impairment that favors escape by immune surveillance. While lots of studies have revealed that tumor-derived EVs can deliver parental materials to adjacent cells and contribute to oncogenic reprogramming, their functionality in energy metabolism is not well addressed. In this study, we established prostate cancer cells PC3-AcT resistant to cellular death in an acidic culture medium driven by lactic acid. Quantitative proteomics between EVs derived from PC-3 and PC-3AcT cells identified 935 confident EV proteins. According to cellular adaptation to lactic acidosis, we revealed 159 regulated EV proteins related to energy metabolism, cellular shape, and extracellular matrix. These EVs contained a high abundance of glycolytic enzymes. In particular, PC-3AcT EVs were enriched with apolipoproteins including apolipoprotein B100 (APOB). APOB on PC-3AcT EVs could facilitate their endocytic uptake depending on low density lipoprotein receptor of recipient PC-3 cells, encouraging increases of cellular proliferation and survival in acidic culture media via increased activity and expression of hexokinases and phosphofructokinase. The activation of recipient PC-3 cells can increase glucose consumption and ATP generation, representing an acquired metabolic reprogramming into the Warburg phenotype. Our study first revealed that EVs derived from prostate cancer cells could contribute to energy metabolic reprogramming and that the acquired metabolic phenotypic transition of recipient cells could favor cellular survival in tumor microenvironment.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100944"},"PeriodicalIF":6.1,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634291","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":"Ca²⁺-triggered (de)ubiquitination events in synapses.","authors":"Sofia Ainatzi, Svenja V Kaufmann, Ivan Silbern, Svilen V Georgiev, Sonja Lorenz, Silvio O Rizzoli, Henning Urlaub","doi":"10.1016/j.mcpro.2025.100946","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100946","url":null,"abstract":"<p><p>Neuronal communication relies on neurotransmitter release from synaptic vesicles (SVs), whose dynamics are controlled by Ca²⁺-dependent pathways, as many thoroughly studied phosphorylation cascades. However, little is known about other post-translational modifications, as ubiquitination. To address this, we analysed resting and stimulated synaptosomes (isolated synapses) by quantitative mass spectrometry. We identified more than 5,000 ubiquitination sites on ∼2,000 proteins, the majority of which participate in SV recycling processes. Several proteins showed significant changes in ubiquitination in response to Ca²⁺ influx, with the most pronounced changes in CaMKIIα and the clathrin adaptor protein AP180. To validate this finding, we generated a CaMKIIα mutant lacking the ubiquitination target site (K291) and analysed it both in neurons and non-neuronal cells. K291 ubiquitination, close to an important site for CaMKIIα autophosphorylation (T286), influences the synaptic function of this kinase. We suggest that ubiquitination in response to synaptic activity is an important regulator of synaptic function.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100946"},"PeriodicalIF":6.1,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634288","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":"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":"https://doi.org/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 (CVs), DIA-MS exhibited greater accuracy in identifying true drug targets and stronger dose-response correlation in protein targets peptides. 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":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Hunt lab weighs in on mass spectrometry-based analysis of protein post-translational modifications.","authors":"Joshua J Coon, Jarrod A Marto, John E P Syka, Forest M White","doi":"10.1016/j.mcpro.2025.100943","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100943","url":null,"abstract":"<p><p>Protein post-translational modifications have traditionally been challenging to identify due to their dynamic regulation and typically low stoichiometry. Methods for phosphopeptide enrichment from complex proteomes developed in the Hunt lab in the late 1990's and early 2000's launched the field of phosphoproteomics, the large-scale analysis of protein phosphorylation sites. To improve phosphopeptide tandem mass spectra and address the further challenge of identifying other labile PTMs such as glycosylation or tyrosine sulfation, the Hunt lab invented and disseminated electron transfer dissociation (ETD), a novel method for peptide and protein fragmentation. Here we provide a brief historical accounting of these discoveries and their ensuing applications.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100943"},"PeriodicalIF":6.1,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625469","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}