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 Pasen, 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":"https://doi.org/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 either 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 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 on the antigenic landscape of the host human cell system, likely reflecting metabolic changes occurred in the infected cells.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100937"},"PeriodicalIF":6.1,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"https://doi.org/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 (MS) is currently a method of choice for direct system-wide identification of cellular immunopeptidome, 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 (DDA) increased HLA-I peptide detection by up to 50%, here, we aimed to evaluate the performance of data-independent acquisition (DIA) in combination with 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 3,426 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 7,742 peptides across 5 conditions and demonstrated that diaPASEF achieves high quantitative accuracy up to 4-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":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic analysis of human follicular fluid-derived exosomes reveals that insufficient folliculogenesis in aging women is associated with infertility.","authors":"Zhen Liu, Qilin Zhou, Jun Zan, Jingyan Tian, Yangzhuohan Zhang, Fanggui Wu, Huan Zhao, Qianwen Peng, Shangjie Liu, Qianjun Chen, Endong Liu, Zhengdong Liao, Pengfei Zou, Lin Mei, Wen Wang, Sen Dong, Luo Niu, Shengda Wu, Liangge He, Xiaoyi Zhou, Yanbo Jin, Panpan Li, Sheng Yang","doi":"10.1016/j.mcpro.2025.100930","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100930","url":null,"abstract":"<p><p>Although the risk of female infertility increases with advancing age, the underlying mechanisms remain unknown. Exosomes in follicular fluid are suggested to regulate folliculogenesis and influence oocyte quality, potentially playing a critical role in age-related infertility. Elucidating their content could enhance the understanding of the molecular mechanisms associated with female aging-induced infertility. In this study, we explored the proteomic profiles of exosomes derived from human follicular fluid to identify protein signatures associated with infertility in both young and aging women. Despite the lack of significant differences in the morphology and particle size of follicular fluid-derived exosomes between the two groups, proteomic analysis revealed a distinct pattern of differentially expressed proteins (DEPs). DEPs associated with B-cell activation, pathogen invasion, and disrupted metabolic processes were significantly more highly expressed in the aging group than in the young group, indicating their involvement in age-related infertility. In vivo experiments demonstrated that the application of exosomes, particularly those derived from young female mice, facilitated the successful maturation of follicles. Key exosomal proteins, including ENO1, HSP90B1, fetuin-B, C7, and APOC4, were found to be associated with follicular maturation. Furthermore, the PI3K/AKT signaling pathway, which is known to be related to folliculogenesis, was activated by the application of exosomes in aging female mice. This study provides novel insights into the aging-associated protein signatures of follicular fluid-derived exosomes and their potential role in infertility. These findings suggest that aging-related protein signatures in exosomes could contribute to the treatment of age-related infertility.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100930"},"PeriodicalIF":6.1,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537412","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":"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":"https://doi.org/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: (i) 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; (ii) 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 (iii) 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 facilitated by EVs a","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100935"},"PeriodicalIF":6.1,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"What have Data Standards ever done for us?","authors":"S E Orchard","doi":"10.1016/j.mcpro.2025.100933","DOIUrl":"https://doi.org/10.1016/j.mcpro.2025.100933","url":null,"abstract":"<p><p>The Human Proteome Organization (HUPO) Proteomics Standards Initiative (PSI) has been successfully developing guidelines, data formats, and controlled vocabularies for both the field of molecular interaction and that of mass spectrometry for more than 20 years. This review explores some of the ways that the proteomics community has benefitted from the development of community standards and takes a look at some of the tools and resources that have been improved or developed as a result of the work of the HUPO-PSI.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100933"},"PeriodicalIF":6.1,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143537421","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":"Integrated multiomics reveals alterations in paucimannose and complex type N-glycans in cardiac tissue of COVID-19 patients.","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":"https://doi.org/10.1016/j.mcpro.2025.100929","url":null,"abstract":"<p><p>Coronavirus infectious disease 19 (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 COVID-19 patients. 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":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","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":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","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":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"How to deal with internal fragment ions?","authors":"Arthur Grimaud, Masa Babovic, Frederik Haugaard Holck, Ole N Jensen, Veit Schwämmle","doi":"10.1016/j.mcpro.2024.100896","DOIUrl":"https://doi.org/10.1016/j.mcpro.2024.100896","url":null,"abstract":"<p><p>Tandem mass spectrometry of peptides and proteins generates mass spectra of their gas-phase fragmentation product ions, including N-terminal, C-terminal, and internal fragment ions. Whereas N- and C-terminal ions are routinely assigned and identified using computational methods, internal fragment ions are often difficult to annotate correctly. They become particularly relevant for long peptides and full proteoforms where the peptide backbone is more likely to be fragmented multiple times. Internal fragment ions potentially offer tremendous information regarding amino acid sequences and positions of post-translational modifications of peptides and intact proteins. However, their practical application is challenged by the vast number of theoretical internal fragments that exist for long amino acid sequences, leading to a high risk of false-positive annotations. We analyze the mass spectral contributions of internal fragment ions in spectra from middle-down and top-down experiments and introduce a novel graph-based annotation approach designed to manage the complexity of internal fragments. Our graph-based representation allows us to compare multiple candidate proteoforms in a single graph, and to assess different candidate annotations in a fragment ion spectrum. We demonstrate cases from middle-down and top-down data where internal ions enhance amino acid sequence coverage of polypeptides and proteins and accurate localization of post-translational modifications. We conclude that our graph-based method provides a general approach to process complex tandem mass spectra, enhance annotation of internal fragment ions, and improve proteoform sequencing and characterization by mass spectrometry.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100896"},"PeriodicalIF":6.1,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425850","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":"HEXB Drives Raised Paucimannosylation in Colorectal Cancer and Stratifies Patient Risk.","authors":"Rebeca Kawahara, Liisa Kautto, Naaz Bansal, Priya Dipta, The Huong Chau, Benoit Liquet-Weiland, Seong Beom Ahn, Morten Thaysen-Andersen","doi":"10.1016/j.mcpro.2025.100927","DOIUrl":"10.1016/j.mcpro.2025.100927","url":null,"abstract":"<p><p>Noninvasive prognostic markers are needed to improve the survival of colorectal cancer (CRC) patients. Toward this goal, we applied untargeted systems glycobiology approaches to snap-frozen and formalin-fixed paraffin-embedded tumor tissues and peripheral blood mononuclear cells from CRC patients spanning different disease stages and matching controls to faithfully uncover molecular changes associated with CRC. Quantitative glycomics and immunohistochemistry revealed that noncanonical paucimannosidic N-glycans are elevated in CRC tumors relative to normal adjacent tissues. Cell origin-focused glycoproteomics enabled using the well-curated Human Protein Atlas combined with immunohistochemistry of CRC tumor tissues recapitulated these findings and indicated that the paucimannosidic proteins were in part from tumor-infiltrating monocytes (e.g., MPO, AZU1) and of CRC cell origin (e.g., LGALS3BP, PSAP). Biosynthetically explaining these observations, N-acetyl-β-D-hexosaminidase (Hex) subunit β (HEXB) was found to be overexpressed in CRC tissues relative to normal adjacent colorectal tissues and colocalization and enzyme inhibition studies confirmed that HEXB facilitates paucimannosidic protein biosynthesis in CRC cells. Employing a sensitive, quick, and robust enzyme activity assay, we then showed that Hex activity was elevated in plasma and peripheral blood mononuclear cells from patients with advanced CRC relative to controls and those with early-stage disease. Surveying a large donor cohort, the plasma Hex activity was found to be raised in CRC patients relative to normal controls and correlated with the 5-year survival of CRC patients indicating that elevated plasma Hex activity is a potential disease risk marker for patient outcome. Our glycoproteomics-driven findings open avenues for better prognostication and disease risk stratification in CRC.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100927"},"PeriodicalIF":6.1,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143414100","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":"Identifying Receptor Kinase Substrates Using an 8000 Peptide Kinase Client Library Enriched for Conserved Phosphorylation Sites.","authors":"Daewon Kim, Gabriel Lemes Jorge, Chunhui Xu, Lingtao Su, Sung-Hwan Cho, Nagib Ahsan, Dongqin Chen, Lijuan Zhou, Marina A Gritsenko, Mowei Zhou, Jinrong Wan, Ljiljana Pasa-Tolic, Dong Xu, Laura E Bartley, Jay J Thelen, Gary Stacey","doi":"10.1016/j.mcpro.2025.100926","DOIUrl":"10.1016/j.mcpro.2025.100926","url":null,"abstract":"<p><p>In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The kinase-client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including integrin-linked kinase 4, for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B-like protein and G protein alpha subunit 1, which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates calcineurin B-like protein and G protein alpha subunit 1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"100926"},"PeriodicalIF":6.1,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143382916","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}