Molecular & Cellular Proteomics最新文献

{"title":"Nucleolar proteomics revealed the regulation of RNA exosome localization by MTR4.","authors":"Yaqian Zhang, Guangzhen Jiang, Ke Wang, Minjie Hong, Xinya Huang, Xiangyang Chen, Xuezhu Feng, Shouhong Guang","doi":"10.1016/j.mcpro.2025.101031","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101031","url":null,"abstract":"<p><p>The nucleolus is the largest membrane-less organelle within the nucleus and plays critical roles in regulating the cell cycle, senescence, and stress responses. The RNA exosome is a multiprotein ribonucleolytic complex involved in RNA processing and degradation in the cytoplasm, the nucleus, and the nucleolus. Previous studies have shown that the subcellular localization of the RNA exosome is crucial for its function. However, the mechanism that regulates its spatial distribution remains largely unexplored. In this study, we identified the nuclear RNA helicase MTR4 as a regulator of the RNA exosome localization through nucleolar quantitative proteomics technology. Immunostaining and fluorescence tagging confirmed that the depletion of MTR4 resulted in the translocation of the RNA exosome subunits from the nucleolus to the nucleoplasm. Notably, the translocation is specifically regulated by MTR4 and does not depend on other cofactors of the MTR4-containing TRAMP, PAXT, and NEXT complexes. The nucleolar accumulation of exosome subunits mutually depends on other exosome subunits. Additionally, actinomycin D treatment, which inhibits transcription, induced the RNA exosome to translocate from the nucleolus to the nucleoplasm, likely through the regulation of nucleolar MTR4 levels. These findings uncover a regulatory mechanism that modulates the localization of the RNA exosome within the nucleolus.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101031"},"PeriodicalIF":6.1,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144619082","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 Exploration of the Immunopeptidome of a Pancreatic Cancer Cell Line: Implications for Clinical Immunopeptidomics and Immunotherapy.","authors":"Mohammadreza Dorvash, Patricia T Illing, Nathan P Croft, Sri H Ramarathinam, Anthony W Purcell","doi":"10.1016/j.mcpro.2025.101030","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101030","url":null,"abstract":"<p><p>Pancreatic ductal adenocarcinoma (PDAC), with its devastating prognosis and limited treatment options, demands innovative therapeutic strategies. T-cell-based immunotherapy has shown promise for many cancers, including PDAC, but is limited by our knowledge of the breadth of cancer-specific T-cell epitopes available. Thus, a comprehensive assessment of the immunopeptidome of PDAC is essential to pave the way for the effective design of immunotherapy and related interventions. In this study, we immunoaffinity purified Human Leukocyte Antigen (HLA) class I-bound peptides from the Panc1 cell line grown in the absence and presence of cytokine stimulation. These peptides were subjected to an off-line high-pH reversed-phase (HPH-RP) fractionation prior to data acquisition. We demonstrate that HPH-RP fractionation followed by data-dependent acquisition (DDA) is a relatively simple and reliable technique that expands the depth of coverage of the PDAC immunopeptidome, allowing the identification of over 22,500 canonical HLA-bound peptides. In addition, the complementary separation by HPH-RP improved the identification confidence, particularly in the case of co-fragmenting precursors in data-independent acquisition (DIA) workflows. This strategy facilitated the identification of a high number of cancer-testis antigen- (CTA-) derived immunopeptides. However, given that fractionation is typically associated with an adsorptive loss, it is impractical to apply HPH-RP on often minuscule clinical specimens and biopsies. Thus, we explored the feasibility of immunopeptidome analysis with cellular inputs as low as 1 million cells (equivalent to approximately 1 mg of tissue) using either a ZenoSWATH DIA interpreted using a spectral library derived from the HPH-RP strategy, or an optimised DDA workflow on the SCIEX ZenoToF 7600 system. Both of these approaches enabled robust detection of CTA-derived and other potentially clinically actionable immunopeptides even at the lowest cellular inputs. We discuss the relative merits of both acquisition strategies and how they can form the basis for future clinical translational immunopeptidomics approaches to screen tumor antigen presentation in low cellular input PDAC biopsies and provide new opportunities for target identification in immune-based therapies. Data are available via the ProteomeXchange with identifiers PXD054360 and PXD054417.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101030"},"PeriodicalIF":6.1,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608830","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":"Nup93-Mediated RNA Alternative Splicing Associated with Diabetic Atherosclerosis.","authors":"Xiaojing Yuan, Qilun Zhang, Jie Li, Zichen Zhang, Shandong Ye, Wei Wang, Wan Zhou","doi":"10.1016/j.mcpro.2025.101029","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101029","url":null,"abstract":"<p><p>Atherosclerosis, a life-threatening complication of diabetes mellitus (DM), significantly increases the mortality risk among diabetic patients. Vascular smooth muscle cells (VSMCs) not only constitute the core of atherosclerotic lesions but also serve as primary components of plaques. Although diabetes expedites this transformation process, the specific mechanism remains elusive. Traditional proteomic approaches that analyse average signals of all cells overlook the importance of spatial information, even though different cells within the same tissue exhibit distinct molecular characteristics during various stages of atherosclerosis progression. In this study, we employed spatial proteomic technology to comprehensively analyse proteins in vascular smooth muscle tissues and atherosclerotic plaques obtained from a mice model of atherosclerosis and DM complicated with arteriosclerosis. We also employed RNA sequencing technology to further investigate the changes in RNA alternative splicing in atherosclerosis and DM complicated with arteriosclerosis cell models. Our finding revealed the reduced expression of Nup93 within VSMCs under combined high glucose and ox-LDL stimulation, mimicking diabetic atherosclerotic stress. This reduction impairs the nuclear import of splicing regulators SRSF1 and SRSF3, leading to abnormal alternative splicing of SerpinE2, which in turn enhances its mRNA stability and promotes VSMCs proliferation. These results reveal a novel mechanistic axis whereby diabetic atherosclerotic stress drives VSMCs dysfunction through Nup93-mediated splicing dysregulation, offering new molecular targets for the treatment of diabetes-associated atherosclerosis.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101029"},"PeriodicalIF":6.1,"publicationDate":"2025-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144600978","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":"Extracellular vesicles bearing vimentin drive epithelial-mesenchymal transition.","authors":"Sepideh Parvanian, Leila S Coelho-Rato, Michael Santos Silva, Giulia Sultana, Arun P Venu, Pallavi Vilas Devre, Mayank Kumar Modi, John E Eriksson","doi":"10.1016/j.mcpro.2025.101028","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101028","url":null,"abstract":"<p><p>Epithelial-mesenchymal transition (EMT) is a key biological process in physiological and pathological conditions, spanning development, wound healing, and cancer. Vimentin, a key cytoskeletal intermediate filament (IF) protein, is an established intracellular determinant of EMT. Recently, extracellular vimentin has also emerged with important functions, and we demonstrated that vimentin from fibroblast-derived extracellular vesicles (EVs) promotes wound healing. Building on these findings, we explored whether extracellular vimentin regulates EMT. We employed fibroblast-derived EVs to assess their EMT-driving capacity. Using co-culture models and EV treatments from wild-type and vimentin-knockout fibroblasts, we observed that fibroblasts induce an EMT phenotype in epithelial cells, marked by elevated mesenchymal markers and reduced epithelial markers. EVs from vimentin-deficient fibroblasts showed a decreased EMT-inducing capacity and failed to stimulate cell cover closure, underscoring vimentin's critical role in orchestrating these processes. Co-culturing epithelial cells with wild-type fibroblasts mirrored these outcomes, while vimentin-deficient fibroblasts produced similarly poor EMT induction. Proteomic profiling revealed that wild-type EVs contained an enriched set of EMT-associated proteins, including those involved in cytoskeletal organization, cell adhesion, and EMT-regulating signaling pathways. Notably, these proteins, such as fibronectin and N-cadherin, were significantly diminished in vimentin-deficient EVs. Moreover, we identified over 600 additional proteins uniquely present in WT-derived EVs, with enrichment in key biological processes like wound healing and cell migration. These findings demonstrate that vimentin-positive EVs drive EMT by transmitting a specific protein cargo that supports EMT-related cellular changes. The vimentin-positive EV proteome will help understand EMT mechanisms and develop targeted therapies for pathological conditions related to abnormal EMT.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101028"},"PeriodicalIF":6.1,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144575912","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":"EGFR phosphorylates and associates with EFNB1 to regulate cell adhesion to fibronectin.","authors":"Ana I Osornio-Hernández, François J M Chartier, Tim L Schuehle, Sara L Banerjee, Sabine Elowe, Patrick Laprise, Andrew Freywald, Mélanie Laurin, Nicolas Bisson","doi":"10.1016/j.mcpro.2025.101027","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101027","url":null,"abstract":"<p><p>Ephrin-Bs (EFNB1-3) are ligands for members of the largest subfamily of receptor tyrosine kinases (RTKs) in humans, the EPH receptors. Interestingly, ephrin-Bs are transmembrane proteins that may also act as receptors themselves upon EPH binding, activating so-called reverse signaling pathways that are critical for multiple cellular processes. Although a number of ephrin-B signaling effectors have been identified, the molecular mechanisms underlying ephrin-B-driven cellular processes remain unresolved, suggesting that multiple signaling effectors are yet to be discovered. Here, we employed proximity labeling proteomics to delineate the proximity network of EFNB1 in steady state and under active reverse signaling conditions. This allowed us to identify 90 uncharacterized EFNB1 proximity partners, from which we could distinguish three main groups: EPH receptor stimulation-dependent, stimulation-independent and negatively modulated by EPH receptor stimulation. We further investigated the functional relationship between EFNB1 and one of the candidates identified, the epidermal growth factor receptor (EGFR). We found that EFNB1 and EGFR associate in cells and showed that the formation of this complex relies on EFNB1's PDZ-binding motif (PBM). Strikingly, we demonstrate that EGFR directly phosphorylates tyrosine residues within EFNB1's PBM, which results in the disruption of the EFNB1-EGFR complex. Furthermore, we show that the EFNB1-EGFR association is required for EFNB1-dependent cell adhesion to fibronectin. Taken together, our results shed light on a functional relationship between EFNB1 and EGFR.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101027"},"PeriodicalIF":6.1,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144575911","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":"Proteomic landscape of colorectal cancer derived liver metastasis reveals three distinct phenotypes with specific signalling and enhanced survival.","authors":"Paula Nissen, Nadezhda V Popova, Antonia Gocke, Daniel J Smit, Geoffrey Yuet Mun Wong, Matthew J McKay, Thomas J Hugh, Kerstin David, Hartmut Juhl, Hannah Voß, Jens U Marquardt, Björn Nashan, Hartmut Schlüter, Mark P Molloy, Manfred Jücker","doi":"10.1016/j.mcpro.2025.101026","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101026","url":null,"abstract":"<p><p>Colorectal carcinoma is a major global disease with the second highest mortality rate among carcinomas. The liver is the most common site for metastases which portends a poor prognosis. Nonetheless, considerable heterogeneity of colorectal cancer liver metastases (CRC-LM) exists, evidenced by varied recurrence and survival patterns in patients undergoing curative-intent resection. Our understanding of the basis for this biological heterogeneity is limited. We investigated this by proteomic analysis of 152 CRC-LM obtained from three different medical centres in Germany and Australia using mass spectrometry based differential quantitative proteomics. The proteomics data of the individual cohorts were harmonized through batch-effect correction algorithms to build a large multi-center cohort. Applying ConsensusClusterPlus to the proteome data yielded three distinct CRC-LM phenotypes (referred to as CRLM-SD, CRLM-CA and CRLM-OM). The CRLM-SD (splice-driven) phenotype showed higher abundance of key regulators of alternative splicing as well as extracellular matrix proteins commonly associated with tumour cell growth. The CRLM-CA (complement-associated) phenotype was characterized by a higher abundance of proteins involved in the classical pathway part of the complement system including the membrane attack complex proteins and those with anti-thrombotic activity. The CRLM-OM (oxidative metabolic) phenotype showed higher abundance of proteins involved in various metabolic pathways including amino acids and fatty acids metabolism, which correlated in the literature with advanced proliferation of metastases and increased recurrence. Patients classified as CRLM-OM had a significantly lower overall survival in comparison to CRLM-CA patients. Finally, we identified a set of prognosis-associated biomarkers for each group including EpCAM, CEACAM1, CEACAM5 and CEACAM6 for CRLM-SD, DCN, TIMP3 and OLFM4 for CRLM-CA and FMO3, CES2 and AGXT for CRLM-OM. In summary, the discovery of three proteomic subgroups associated with distinct signalling pathways and survival of the CRC-LM patients provides a novel classification for risk stratification, prognosis and potentially novel therapeutic targets in CRC-LM.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101026"},"PeriodicalIF":6.1,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144567546","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":"Proteome profiling of cerebrospinal fluid and machine learning reveal protein classifiers of two forms of Alzheimer's disease characterized by increased or not altered levels of tau.","authors":"Elisabetta Scalia, Matteo Calligaris, Margot Lo Pinto, Salvatore Castelbuono, Matilda Iemmolo, Vincenzina Lo Re, Giulia Bivona, Tommaso Piccoli, Giulio Ghersi, Simone Dario Scilabra","doi":"10.1016/j.mcpro.2025.101025","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101025","url":null,"abstract":"<p><p>Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that presents with heterogeneous clinical and pathological features, necessitating improved biomarkers for accurate diagnosis and patient stratification. In this study, we applied a data-independent acquisition (DIA)-based proteomics workflow to cerebrospinal fluid (CSF) samples from 138 individuals, including AD patients with high (Aβ+/tau+) or normal (Aβ+/tau-) CSF tau levels, and non-AD controls. Analysis using an Astral mass spectrometer enabled unprecedented proteome depth, identifying 2661 proteins with high data completeness. Comparative proteomic profiling revealed distinct protein signatures for Aβ+/tau+ and Aβ+/tau- subtypes. These findings were validated using an independent internal cohort and further corroborated with publicly available datasets from larger external AD cohorts, demonstrating the robustness and reproducibility of our results. Using machine learning, we identified a panel of 15 protein classifiers that accurately distinguished the two AD subtypes and controls across datasets. Notably, several of these proteins were elevated in the preclinical stage, underscoring their potential utility for early diagnosis and stratification. Together, our results demonstrate the power of DIA proteomics on the Astral platform, combined with machine learning, to uncover subtype-specific biomarkers of AD and support the development of personalized diagnostic strategies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101025"},"PeriodicalIF":6.1,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144553950","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":"Signatures of the systemic effects of a snake venom and antivenom: multiomics profiling of the kidney pathology.","authors":"Alison F A Chaves, Bianca C S C de Barros, Miguel Cosenza-Contreras, Mariana S L C Morone, Ana T A Sachetto, Niko Pinter, Marlene Schmid, Marcelo L Santoro, Oliver Schilling, Solange M T Serrano","doi":"10.1016/j.mcpro.2025.101023","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101023","url":null,"abstract":"<p><p>Animal venoms comprise many toxins that work in concert to break apart the robust homeostatic systems of prey organisms. Conversely, prey organisms actively antagonize each step of envenoming, which displays a complex kinetics involving important changes at molecular, cell, tissue, and organism levels. In this study we explored the mammalian host response to envenoming using proteomics/N-terminomics and phosphoproteomics approaches to evaluate the in vivo effects of Bothrops jararaca venom in the mouse kidney after injection in the thigh muscle (1.6 mg/kg), mimicking a snakebite, and the impact of anti-Bothrops antivenom injected 1 h later (1.6 mg/kg; i.v. tail). For proteomics/N-terminomics, proteins were TMT-labeled, in order to allow for specific (tryptic) and semi-specific searches of MS/MS spectra to assess both global proteome and degradome. We quantified > 7,000 proteins and prominent changes were observed in the kidney tissue, where protein differential abundance was identified after 3, 6 and 24 h, including markers of acute-phase response and injury. Likewise, the N-terminomic analysis revealed a significant impact of venom progressing from 3 h to 24 h, resulting in dysregulated proteolysis, and indicating the activation of host proteases. The protease fingerprint matched legumain and cathepsin profiles. Venom toxins also promoted alteration in the dynamics of phosphorylation, with the activation of kinases. Under the conditions tested, antivenom administration (i) did not reduce the number of differentially abundant proteins and inflammation markers, (ii) partially attenuated the generation of proteolytic products in envenomed animals, and (iii) directly perturbed the phosphorylation signaling in control animals. Taken together, our findings underscore for the first time the mouse renal response to a protease-rich venom, revealed by the dynamic alteration of protein abundance, protease targets and phosphorylation events, providing new facets of snake venom and antivenom systemic effects, which are important for the development of new therapies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101023"},"PeriodicalIF":6.1,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528914","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 Roadmap for Improving Reliability and Data Sharing in Crosslinking Mass Spectrometry.","authors":"Juri Rappsilber, James Bruce, Colin Combe, Stephen Fried, Andrea Graziadei, Albert J R Heck, Claudio Iacobucci, Alexander Leitner, Karl Mechtler, Petr Novak, Francis O'Reilly, David C Schriemer, Andrea Sinz, Florian Stengel, Konstantinos Thalassinos","doi":"10.1016/j.mcpro.2025.101024","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101024","url":null,"abstract":"<p><p>Crosslinking Mass Spectrometry (MS) can uncover protein-protein interactions and provide structural information on proteins in their native cellular environments. Despite its promise, the field remains hampered by inconsistent data formats, variable approaches to error control, and insufficient interoperability with global data repositories. Recent advances, especially in false discovery rate (FDR) models and pipeline benchmarking, show that Crosslinking MS data can reach a reliability that matches the demand of integrative structural biology. To drive meaningful progress, however, the community must agree on error estimation, open data formats, and streamlined repository submissions. This perspective highlights these challenges, clarifies remaining barriers, and frames practical next steps. Successful field harmonisation will enhance the acceptance of Crosslinking MS in the broader biological community and is critical for the dependability of the data, no matter where it is produced.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101024"},"PeriodicalIF":6.1,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144528913","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 Methanolic Urea-enhanced Protein Extraction Enabling the Largest Bacterial Phosphorylation Resource.","authors":"Pei-Shan Wu, Ting-An Chen, Bo-Yu Chen, Yasushi Ishihama, Miao-Hsia Lin","doi":"10.1016/j.mcpro.2025.101019","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.101019","url":null,"abstract":"<p><p>Mass spectrometry (MS)-based phosphoproteomics analysis is a powerful approach for elucidating the regulatory roles of protein phosphorylation across all domain of life. However, bacterial phosphoproteomics still faces significant technical challenges due to the extremely low substoichiometry of phosphorylation evens and the structural complexity of bacterial cell envelopes, which impede efficient cell lysis, protein recovery and purity. To address these obstacles, we developed Methanolic Urea-enhanced Protein Extraction (MUPE), a streamlined, detergent-free, solvent-based method that leverages the amphiphilic nature of methanol and the chaotropic properties of urea to enhance protein yield and lysis efficiency. Furthermore, MUPE seamlessly integrates with liquid-liquid extraction, enabling efficient protein purification without requiring sample transfer and complex manipulations. This workflow significantly improves phosphoproteome coverage and quantitative accuracy across Gram-positive and Gram-negative bacteria, while minimizing sample input requirements. Our datasets substantially expand the known landscape of bacterial O-phosphorylation, revealing distinct phosphorylation preferences within bacterial signaling networks. Application of MUPE to Listeria monocytogenes under bile insult revealed extensive phosphorylation changes independent of protein expression, highlighting phosphorylation as a rapid and dynamic regulatory mechanism. Collectively, MUPE provides a robust and scalable platform for bacterial phosphoproteomic studies, advancing our understanding of phosphosignaling in the context of bacterial physiology and pathogenesis.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101019"},"PeriodicalIF":6.1,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144506830","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}