Pub Date : 2025-07-31DOI: 10.1016/j.jprot.2025.105511
Diogo B. Lima , Max Ruwolt , Marlon D.M. Santos , Ke Pu , Fan Liu , Paulo C. Carvalho
We present Q2C, an open-source software designed to streamline mass spectrometer queue management and assess performance based on quality control metrics. Q2C provides a fast and user-friendly interface to visualize projects queues, manage analysis schedules and keep track of samples that were already processed. Our software includes analytical tools to ensure equipment calibration and provides comprehensive log documentation for machine maintenance, enhancing operational efficiency and reliability. Additionally, Q2C integrates with Google™ Cloud, allowing users to access and manage the software from different locations while keeping all data synchronized and seamlessly integrated across the system. For multi-user environments, Q2C implements a write-locking mechanism that checks for concurrent operations before saving data. When conflicts are detected, subsequent write requests are automatically queued to prevent data corruption, while the interface continuously refreshes to display the most current information from the cloud storage. Finally, Q2C, a demonstration video, and a user tutorial are freely available for academic use at https://github.com/diogobor/Q2C. Data are available from the ProteomeXchange consortium (identifier PXD055186).
Significance
Q2C addresses a critical gap in mass spectrometry facility management by unifying sample queue management with instrument performance monitoring. It ensures optimal instrument utilization, reduces turnaround times, and enhances data quality by dynamically prioritizing and routing samples based on analysis type and urgency. Unlike existing tools, Q2C integrates queue control and QC in a single platform, maximizing operational efficiency and reliability.
{"title":"Q2C: A software for managing mass spectrometry facilities","authors":"Diogo B. Lima , Max Ruwolt , Marlon D.M. Santos , Ke Pu , Fan Liu , Paulo C. Carvalho","doi":"10.1016/j.jprot.2025.105511","DOIUrl":"10.1016/j.jprot.2025.105511","url":null,"abstract":"<div><div>We present Q2C, an open-source software designed to streamline mass spectrometer queue management and assess performance based on quality control metrics. Q2C provides a fast and user-friendly interface to visualize projects queues, manage analysis schedules and keep track of samples that were already processed. Our software includes analytical tools to ensure equipment calibration and provides comprehensive log documentation for machine maintenance, enhancing operational efficiency and reliability. Additionally, Q2C integrates with Google™ Cloud, allowing users to access and manage the software from different locations while keeping all data synchronized and seamlessly integrated across the system. For multi-user environments, Q2C implements a write-locking mechanism that checks for concurrent operations before saving data. When conflicts are detected, subsequent write requests are automatically queued to prevent data corruption, while the interface continuously refreshes to display the most current information from the cloud storage. Finally, Q2C, a demonstration video, and a user tutorial are freely available for academic use at <span><span>https://github.com/diogobor/Q2C</span><svg><path></path></svg></span>. Data are available from the ProteomeXchange consortium (identifier PXD055186).</div></div><div><h3>Significance</h3><div>Q2C addresses a critical gap in mass spectrometry facility management by unifying sample queue management with instrument performance monitoring. It ensures optimal instrument utilization, reduces turnaround times, and enhances data quality by dynamically prioritizing and routing samples based on analysis type and urgency. Unlike existing tools, Q2C integrates queue control and QC in a single platform, maximizing operational efficiency and reliability.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105511"},"PeriodicalIF":2.8,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144768734","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}
Pub Date : 2025-07-29DOI: 10.1016/j.jprot.2025.105510
Qiaozhen Xu , Feng Tian , Xinyun Wang , Juanqi Lian , Xiaowei Zhang , Xiangmin Lin , Yanling Liu
Biofilms play a pivotal role in the survival and persistence of microorganisms, endowing them with heightened resistance to environmental stressors and antimicrobial agents. The EamB protein, which encodes an inner membrane transporter, acted as a negative regulator of biofilm formation, and the gene eamB deletion in the pathogen Aeromonas hydrophila LP-2 resulted in a significant increase in biofilm formation. Proteomic analysis revealed a total of 616 differentially abundant proteins between the ΔeamB and wild-type (WT) strains, with 308 downregulated and 308 upregulated. RT-qPCR was employed to verify the stability and accuracy of the proteomics data. Bioinformatic analysis indicated that EamB is involved in critical bacterial biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Biofilm formation assays further revealed that supplementation with exogenous lysine significantly inhibited biofilm formation in the ΔeamB strain, conversely, exogenous cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings demonstrated that EamB may modulate bacterial biofilm formation in A. hydrophila through the regulation of amino acid metabolism. This finding provides novel insights into the regulatory mechanism underlying biofilm formation and highlights potential targets for the development of future antibacterial strategies.
Significance statement
This study elucidates the critical role of the eamB gene in Aeromonas hydrophila, a significant aquatic pathogen, by demonstrating its impact on biofilm formation and physiological traits. Through comparative proteomic analysis, we identified 616 differentially abundant proteins in the ΔeamB mutant, revealing its involvement in key metabolic pathways such as amino acid metabolism, flagellar assembly, and fatty acid degradation. Notably, eamB deletion enhanced biofilm formation, while exogenous amino acids like cysteine and O-acetylserine obviously increased biofilm formation in the ΔeamB strain. These findings highlight EamB as a regulator of biofilm formation, offering novel molecular insights into bacterial pathogenicity. This research advances our understanding of biofilm-associated antibiotic resistance and provides potential targets for developing strategies to mitigate infections caused by A. hydrophila in aquaculture and public health.
{"title":"Proteomics reveals the role of the EamB transporter from Aeromonas hydrophila LP-2 in biofilm formation","authors":"Qiaozhen Xu , Feng Tian , Xinyun Wang , Juanqi Lian , Xiaowei Zhang , Xiangmin Lin , Yanling Liu","doi":"10.1016/j.jprot.2025.105510","DOIUrl":"10.1016/j.jprot.2025.105510","url":null,"abstract":"<div><div>Biofilms play a pivotal role in the survival and persistence of microorganisms, endowing them with heightened resistance to environmental stressors and antimicrobial agents. The EamB protein, which encodes an inner membrane transporter, acted as a negative regulator of biofilm formation, and the gene <em>eamB</em> deletion in the pathogen <em>Aeromonas hydrophila</em> LP-2 resulted in a significant increase in biofilm formation. Proteomic analysis revealed a total of 616 differentially abundant proteins between the <em>ΔeamB</em> and wild-type (WT) strains, with 308 downregulated and 308 upregulated. RT-qPCR was employed to verify the stability and accuracy of the proteomics data. Bioinformatic analysis indicated that EamB is involved in critical bacterial biological processes, including flagellar assembly, amino acid metabolism, and fatty acid degradation. Biofilm formation assays further revealed that supplementation with exogenous lysine significantly inhibited biofilm formation in the <em>ΔeamB</em> strain, conversely, exogenous cysteine and <em>O</em>-acetylserine obviously increased biofilm formation in the <em>ΔeamB</em> strain. These findings demonstrated that EamB may modulate bacterial biofilm formation in <em>A. hydrophila</em> through the regulation of amino acid metabolism. This finding provides novel insights into the regulatory mechanism underlying biofilm formation and highlights potential targets for the development of future antibacterial strategies.</div></div><div><h3>Significance statement</h3><div>This study elucidates the critical role of the <em>eamB</em> gene in <em>Aeromonas hydrophila</em>, a significant aquatic pathogen, by demonstrating its impact on biofilm formation and physiological traits. Through comparative proteomic analysis, we identified 616 differentially abundant proteins in the <em>ΔeamB</em> mutant, revealing its involvement in key metabolic pathways such as amino acid metabolism, flagellar assembly, and fatty acid degradation. Notably, <em>eamB</em> deletion enhanced biofilm formation, while exogenous amino acids like cysteine and <em>O</em>-acetylserine obviously increased biofilm formation in the Δ<em>eamB</em> strain. These findings highlight EamB as a regulator of biofilm formation, offering novel molecular insights into bacterial pathogenicity. This research advances our understanding of biofilm-associated antibiotic resistance and provides potential targets for developing strategies to mitigate infections caused by <em>A. hydrophila</em> in aquaculture and public health.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105510"},"PeriodicalIF":2.8,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144760414","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jprot.2025.105507
John Oluwafemi Teibo , Roberta Maraninchi Silveira , Virginia Campos Silvestrini , Izadora Archiolli , Ana Paula Masson , Beatriz Pereira de Morais , Dayane Schmidt , Matheus Henrique dos Santos , Germano Aguiar Ferreira , Carolina Hassibe Thomé , Dominic Helm , Raja Sekhar Nirujogi , Dairo Renato Alessi , Virginia Picanço-Castro , Lucas Eduardo Botelho de Souza , Vitor Marcel Faça
Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of the field of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation anti-CD19 CAR construct, which allowed us to analyse early proteomic changes that are crucial for comprehending the signalling pathways and mechanism of action of this CAR-T cell. SILAC-heavy tagged Raji B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were acquired via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in about 8800 protein identifications at 1 % FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target Raji B-cells exhibited more significant alterations. Effector proteins, namely CD247, CD28, DAP, LCK, p38 MAPK, and CASP3, were validated, as they have critical roles in antigen presentation, T-cell activation, and apoptosis. Pharmacological inhibition of LCK using Dasatinib further suggested its pivotal role in early CAR-T signalling. This study led us to identify proteins that function as molecular effectors of anti-CD19 CAR-T cell therapy during the initial phases of CAR-T-target cell engagement, advancing our knowledge of the mechanism and signalling pathways that will support CAR-T cell development.
Significance
Chimeric antigen receptor T-cell (CAR-T cell) therapy is state-of-the-art in cell and immunotherapy. Determining important players in cellular communication and signalling mediated by membranes and intracellular proteins requires understanding the connection between tumours and modified cells. We employed global proteomics in this study to better grasp the functional protein networks using a high-sensitivity mass spectrometric platform for protein identification and quantification. We identified proteins as molecular effectors of anti-CD19 CAR-T cell treatment during the early stages of CAR-T-target cell interaction. Our understanding of the mechanism and signalling pathways will promote the development of new CAR constructs and improve the efficacy and ability to overcome the resistance of this innovative cancer treatment strategy, which will advance the identification of adjuvant molecules for the regulation of CAR-T responses.
{"title":"Proteomics analysis reveal early event molecular effectors of anti-CD19 CAR-T cell therapy in hematological cancer","authors":"John Oluwafemi Teibo , Roberta Maraninchi Silveira , Virginia Campos Silvestrini , Izadora Archiolli , Ana Paula Masson , Beatriz Pereira de Morais , Dayane Schmidt , Matheus Henrique dos Santos , Germano Aguiar Ferreira , Carolina Hassibe Thomé , Dominic Helm , Raja Sekhar Nirujogi , Dairo Renato Alessi , Virginia Picanço-Castro , Lucas Eduardo Botelho de Souza , Vitor Marcel Faça","doi":"10.1016/j.jprot.2025.105507","DOIUrl":"10.1016/j.jprot.2025.105507","url":null,"abstract":"<div><div>Chimeric antigen receptor T-cell (CAR-T) therapy is at the forefront of the field of cell immunotherapy. In this study, we generated an anti-CD19 CAR-Jurkat T cell line using a locally produced second-generation anti-CD19 CAR construct, which allowed us to analyse early proteomic changes that are crucial for comprehending the signalling pathways and mechanism of action of this CAR-T cell. SILAC-heavy tagged Raji B-cells and anti-CD19 CAR-Jurkat T-cells were co-cultured for ten minutes. The proteomic profiles were acquired via DIA methodology on the Orbitrap Astral LC-MS/MS platform. The proteome was extensively covered, resulting in about 8800 protein identifications at 1 % FDR. The effector CAR-Jurkat cells showed proteomic changes involving antigen presentation by CD74. The target Raji B-cells exhibited more significant alterations. Effector proteins, namely CD247, CD28, DAP, LCK, p38 MAPK, and CASP3, were validated, as they have critical roles in antigen presentation, T-cell activation, and apoptosis. Pharmacological inhibition of LCK using Dasatinib further suggested its pivotal role in early CAR-T signalling. This study led us to identify proteins that function as molecular effectors of anti-CD19 CAR-T cell therapy during the initial phases of CAR-T-target cell engagement, advancing our knowledge of the mechanism and signalling pathways that will support CAR-T cell development.</div></div><div><h3>Significance</h3><div>Chimeric antigen receptor T-cell (CAR-T cell) therapy is state-of-the-art in cell and immunotherapy. Determining important players in cellular communication and signalling mediated by membranes and intracellular proteins requires understanding the connection between tumours and modified cells. We employed global proteomics in this study to better grasp the functional protein networks using a high-sensitivity mass spectrometric platform for protein identification and quantification. We identified proteins as molecular effectors of anti-CD19 CAR-T cell treatment during the early stages of CAR-T-target cell interaction. Our understanding of the mechanism and signalling pathways will promote the development of new CAR constructs and improve the efficacy and ability to overcome the resistance of this innovative cancer treatment strategy, which will advance the identification of adjuvant molecules for the regulation of CAR-T responses.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105507"},"PeriodicalIF":2.8,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731928","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jprot.2025.105509
Karina Araujo-Ruiz , Daniel Ignacio López-Flores , María Karla Martínez-Muné , Brenda Yomara García-Sánchez , Carlos J. Ramírez-Flores , Francisco Ernesto Sandoval-Rodríguez , Emmanuel Ríos-Castro , Mónica Edith Mondragón-Castelán , Sirenia González-Pozos , Ricardo Mondragón-Flores
The Toxoplasma gondii cytoskeleton is a highly organized structure essential for parasite motility, replication, and host cell invasion. To identify its components, a highly enriched fraction of tachyzoite cytoskeletons was obtained and quantitatively analyzed by mass spectrometry. We identified 623 proteins classified into 18 functional groups, including 30 IMC proteins, 34 cytoskeleton proteins, and 14 uncharacterized proteins. A comprehensive bioinformatic analysis was conducted to assess protein abundance (fmol), antigenicity, accessibility, interactome, and homology, with the aim of identifying immunogenic targets. Among the top vaccine candidates were -GRA12, IMC1, ROP8, and -IMC4, with ROP8 emerging as the most promising based on epitope prediction. Data are available via ProteomeXchange with identifier PXD063409.
Significance
Toxoplasma gondii represents one of the most virulent and successful parasites in human and veterinary pathogenesis. Since T. gondii is a highly dynamic parasite that depends on its cytoskeleton to invade and disseminate through tissues, knowledge of its cytoskeleton composition is essential for understanding the biological mechanisms involved in parasite-host interactions and for the design of pharmaceutical and vaccination strategies. Quantitative proteomic analysis of the T. gondii cytoskeleton provided new and extensive information on its composition and, through bioinformatics approaches, allowed us to suggest several candidate molecules for future immunoprotective design.
{"title":"Quantitative proteomic analysis of the Toxoplasma gondii cytoskeleton and bioinformatic identification of highly antigenic proteins","authors":"Karina Araujo-Ruiz , Daniel Ignacio López-Flores , María Karla Martínez-Muné , Brenda Yomara García-Sánchez , Carlos J. Ramírez-Flores , Francisco Ernesto Sandoval-Rodríguez , Emmanuel Ríos-Castro , Mónica Edith Mondragón-Castelán , Sirenia González-Pozos , Ricardo Mondragón-Flores","doi":"10.1016/j.jprot.2025.105509","DOIUrl":"10.1016/j.jprot.2025.105509","url":null,"abstract":"<div><div>The <em>Toxoplasma gondii</em> cytoskeleton is a highly organized structure essential for parasite motility, replication, and host cell invasion. To identify its components, a highly enriched fraction of tachyzoite cytoskeletons was obtained and quantitatively analyzed by mass spectrometry. We identified 623 proteins classified into 18 functional groups, including 30 IMC proteins, 34 cytoskeleton proteins, and 14 uncharacterized proteins. A comprehensive bioinformatic analysis was conducted to assess protein abundance (fmol), antigenicity, accessibility, interactome, and homology, with the aim of identifying immunogenic targets. Among the top vaccine candidates were -GRA12, IMC1, ROP8, and -IMC4, with ROP8 emerging as the most promising based on epitope prediction. Data are available <em>via</em> ProteomeXchange with identifier <span><span>PXD063409</span><svg><path></path></svg></span>.</div></div><div><h3>Significance</h3><div><em>Toxoplasma gondii</em> represents one of the most virulent and successful parasites in human and veterinary pathogenesis. Since <em>T. gondii</em> is a highly dynamic parasite that depends on its cytoskeleton to invade and disseminate through tissues, knowledge of its cytoskeleton composition is essential for understanding the biological mechanisms involved in parasite-host interactions and for the design of pharmaceutical and vaccination strategies. Quantitative proteomic analysis of the <em>T. gondii</em> cytoskeleton provided new and extensive information on its composition and, through bioinformatics approaches, allowed us to suggest several candidate molecules for future immunoprotective design.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105509"},"PeriodicalIF":2.8,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144722115","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}
The mechanisms underlying cell polarization are fundamental to biology but remain incompletely understood. This is especially true for hepatocytes, which display a particularly complex polarization that enables the formation of the bile canaliculi (BC) network crucial for liver excretory functions. To identify key proteins involved in hepatocyte polarization, BC formation, structure or function, we employed a proteomic approach comparing the human hepatocyte cell line HepG2 to its sub clone HepG2/C3A known for its markedly greater efficiency in forming mature BCs. Through this analysis, we localized LimA1 and Espin to the BC for the first time, suggesting their important role in this compartment, and confirmed the presence of NHE-RF1. Using a targeted protein repression strategy, we identified E cadherin as essential for the initiation of BC formation, unlike other adherens junction components such as N cadherin or α-catenin. Our findings demonstrate, for the first time, that in the absence of E cadherin, hepatocytes lose the capacity to form BCs.
Significance
This study aims to deepen our understanding of the highly specialized polarization of hepatocytes in relation to bile canaliculus formation. The major finding is the key role of E cadherin in this process, where it appears to be essential for bile canaliculus formation in both 2D and 3D culture models. Additionally, the study led to the identification of several proteins potentially localized to the bile canaliculi, whose functions remain to be elucidated.
{"title":"E cadherin appears to be an essential on/off switch for initiating bile canaliculi formation","authors":"Mireille Chevallet , Thierry Rabilloud , Hélène Diemer , Fabrice Bertile , Alexandra Fuchs , Aurélien Deniaud","doi":"10.1016/j.jprot.2025.105508","DOIUrl":"10.1016/j.jprot.2025.105508","url":null,"abstract":"<div><div>The mechanisms underlying cell polarization are fundamental to biology but remain incompletely understood. This is especially true for hepatocytes, which display a particularly complex polarization that enables the formation of the bile canaliculi (BC) network crucial for liver excretory functions. To identify key proteins involved in hepatocyte polarization, BC formation, structure or function, we employed a proteomic approach comparing the human hepatocyte cell line HepG2 to its sub clone HepG2/C3A known for its markedly greater efficiency in forming mature BCs. Through this analysis, we localized LimA1 and Espin to the BC for the first time, suggesting their important role in this compartment, and confirmed the presence of NHE-RF1. Using a targeted protein repression strategy, we identified E cadherin as essential for the initiation of BC formation, unlike other adherens junction components such as N cadherin or α-catenin. Our findings demonstrate, for the first time, that in the absence of E cadherin, hepatocytes lose the capacity to form BCs.</div></div><div><h3>Significance</h3><div>This study aims to deepen our understanding of the highly specialized polarization of hepatocytes in relation to bile canaliculus formation. The major finding is the key role of E cadherin in this process, where it appears to be essential for bile canaliculus formation in both 2D and 3D culture models. Additionally, the study led to the identification of several proteins potentially localized to the bile canaliculi, whose functions remain to be elucidated.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105508"},"PeriodicalIF":2.8,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718127","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}
Redox regulation has emerged as a key process in cellular signaling. The role of extracellular cell surface redox-sensitive proteins in redox regulation and intracellular communication has been supported by secretion of oxidoreductases that modulate thiol-disulfide switches. Despite these advances, redox-sensitive targets on the cell surface remain little explored. We established a comprehensive redox proteomic workflow using plasma membrane impermeable thiol labeling where we identified 1159 cell surface and extracellular proteins susceptible to oxidation. Treatment with diamide or urate hydroperoxide (HOOU) resulted in 377 and 12 differentially abundant redox-modulated proteins compared to control. Such proteins represent chaperones, adhesion molecules, vesicle-associated proteins, channels, receptors, cytoskeleton, and others, which may play a relevant role in several signaling pathway. Eleven oxidoreductases were redox-modulated by diamide, including members of the protein disulfide isomerase (PDI), peroxiredoxin (PRDX), and quiescin sulfhydryl oxidase (QSOX) families, with a particular focus on PDI TMX3 (TMX3), which provides the first evidence of its secretion in endothelial cells. In conclusion, our findings not only revealed potential redox-sensitive targets on the cell surface but also offer a useful tool for future investigations aiming to analyze redox regulation in the extracellular environment across diverse biological contexts.
Significance
Redox signaling at the cell surface is emerging as a crucial regulator of vascular function, emphasizing its role in cardiovascular disease. However, the extracellular redox proteome remains underexplored because of the complexity of the method. We developed a reproducible workflow combining differential thiol labeling and mass spectrometry to systematically map oxidized extracellular proteins in endothelial cells exposed to oxidants. Hundreds of proteins were identified as redox-sensitive targets. Key functional groups included molecular chaperones, adhesion molecules, vesicle-associated proteins, channels, receptors, and cytoskeleton. This work reveals novel insights into extracellular redox regulation, expands the repertoire of known redox-sensitive proteins, and establishes a versatile platform to investigate redox dynamics at cell surface both in vascular biology and other pathophysiological contexts.
{"title":"Redox proteomics workflow to unveil extracellular targets of oxidation in vascular endothelial cells","authors":"Danielle Fernandes Vileigas, Railmara Pereira da Silva, Bianca Dempsey, Mariana Pereira Massafera, Mikaela Peglow Pinz, Flavia Carla Meotti","doi":"10.1016/j.jprot.2025.105506","DOIUrl":"10.1016/j.jprot.2025.105506","url":null,"abstract":"<div><div>Redox regulation has emerged as a key process in cellular signaling. The role of extracellular cell surface redox-sensitive proteins in redox regulation and intracellular communication has been supported by secretion of oxidoreductases that modulate thiol-disulfide switches. Despite these advances, redox-sensitive targets on the cell surface remain little explored. We established a comprehensive redox proteomic workflow using plasma membrane impermeable thiol labeling where we identified 1159 cell surface and extracellular proteins susceptible to oxidation. Treatment with diamide or urate hydroperoxide (HOOU) resulted in 377 and 12 differentially abundant redox-modulated proteins compared to control. Such proteins represent chaperones, adhesion molecules, vesicle-associated proteins, channels, receptors, cytoskeleton, and others, which may play a relevant role in several signaling pathway. Eleven oxidoreductases were redox-modulated by diamide, including members of the protein disulfide isomerase (PDI), peroxiredoxin (PRDX), and quiescin sulfhydryl oxidase (QSOX) families, with a particular focus on PDI TMX3 (TMX3), which provides the first evidence of its secretion in endothelial cells. In conclusion, our findings not only revealed potential redox-sensitive targets on the cell surface but also offer a useful tool for future investigations aiming to analyze redox regulation in the extracellular environment across diverse biological contexts.</div></div><div><h3>Significance</h3><div>Redox signaling at the cell surface is emerging as a crucial regulator of vascular function, emphasizing its role in cardiovascular disease. However, the extracellular redox proteome remains underexplored because of the complexity of the method. We developed a reproducible workflow combining differential thiol labeling and mass spectrometry to systematically map oxidized extracellular proteins in endothelial cells exposed to oxidants. Hundreds of proteins were identified as redox-sensitive targets. Key functional groups included molecular chaperones, adhesion molecules, vesicle-associated proteins, channels, receptors, and cytoskeleton. This work reveals novel insights into extracellular redox regulation, expands the repertoire of known redox-sensitive proteins, and establishes a versatile platform to investigate redox dynamics at cell surface both in vascular biology and other pathophysiological contexts.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"321 ","pages":"Article 105506"},"PeriodicalIF":2.8,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718128","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}
Pub Date : 2025-07-21DOI: 10.1016/j.jprot.2025.105505
Junmei Zhang , Shanling Cao , Zheng Tian , Zhengzhe Zhang , Yu Zhou , Yixuan Wu , Xuenong Luo , Shuai Wang , Xiaola Guo
Alveolar echinococcosis is a zoonotic disease that poses serious threats to public health. We observed subcutaneous cysts (SCs) of E. multilocularis had fewer protoscoleces (PSCs) compared to intraperitoneal cysts (ICs) at 60 days post-infection. However, the mechanisms underlying the development of E. multilocularis cysts in different tissues remain unclear. In this study, we compared the proteomic profiles of E. multilocularis cysts derived from mice intraperitoneally and subcutaneously infected with PSCs at 30 days post-infection, prior to the development of mature PSCs. Proteomic analysis identified 284 differentially expressed proteins (DEPs) in SCs compared to ICs, with 147 upregulated DEPs and 137 downregulated DEPs. Enzymatic proteins involved in carbohydrate and amino acid metabolism were predominantly upregulated in SCs compared to ICs, whereas proteins associated with protein folding, sorting, a degradation were downregulated. Western blotting analysis confirmed that phosphoenolpyruvate carboxykinase (PEPCK) and fructose-bisphosphate aldolase (FBA) were upregulated, whereas transitional endoplasmic reticulum ATPase (TER ATPase) was downregulated in SCs compared to ICs. The identified DEPs may play crucial roles in shaping the unique characteristics of E. multilocularis cysts. This study offers valuable insights into exploring the mechanisms underlying the occurrence and development of metacestodes.
{"title":"Comparative proteomic analysis of subcutaneous and intraperitoneal cysts of Echinococcus multilocularis","authors":"Junmei Zhang , Shanling Cao , Zheng Tian , Zhengzhe Zhang , Yu Zhou , Yixuan Wu , Xuenong Luo , Shuai Wang , Xiaola Guo","doi":"10.1016/j.jprot.2025.105505","DOIUrl":"10.1016/j.jprot.2025.105505","url":null,"abstract":"<div><div>Alveolar echinococcosis is a zoonotic disease that poses serious threats to public health. We observed subcutaneous cysts (SCs) of <em>E. multilocularis</em> had fewer protoscoleces (PSCs) compared to intraperitoneal cysts (ICs) at 60 days post-infection. However, the mechanisms underlying the development of <em>E. multilocularis</em> cysts in different tissues remain unclear. In this study, we compared the proteomic profiles of <em>E. multilocularis</em> cysts derived from mice intraperitoneally and subcutaneously infected with PSCs at 30 days post-infection, prior to the development of mature PSCs. Proteomic analysis identified 284 differentially expressed proteins (DEPs) in SCs compared to ICs, with 147 upregulated DEPs and 137 downregulated DEPs. Enzymatic proteins involved in carbohydrate and amino acid metabolism were predominantly upregulated in SCs compared to ICs, whereas proteins associated with protein folding, sorting, a degradation were downregulated. Western blotting analysis confirmed that phosphoenolpyruvate carboxykinase (PEPCK) and fructose-bisphosphate aldolase (FBA) were upregulated, whereas transitional endoplasmic reticulum ATPase (TER ATPase) was downregulated in SCs compared to ICs. The identified DEPs may play crucial roles in shaping the unique characteristics of <em>E. multilocularis</em> cysts. This study offers valuable insights into exploring the mechanisms underlying the occurrence and development of metacestodes.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"320 ","pages":"Article 105505"},"PeriodicalIF":2.8,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144698902","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}
Pub Date : 2025-07-19DOI: 10.1016/j.jprot.2025.105503
Giulia Riccomi , Lisa Rosselli , Marinella Marchesi , Federica Guidi , Maria Taloni , Giovanni Ricci , Carmine Pellegrino , Shevan Wilkin
Periodontitis, a chronic inflammatory disease affecting the tooth-supporting structures, is a key indicator of oral health in palaeopathology. While poor oral hygiene, systemic diseases, and genetics are well-established contributors, the dietary impact has often been underestimated. Clinical studies, however, link diets high in fermentable carbohydrates and meat to inflammation. We investigated periodontal disease by analyzing interdental septa in 63 individuals from elite and non-elite groups in pre-Roman Italy (7th–4th centuries BCE), a period of social stratification, intensified agriculture, and increased cereal consumption. Macroscopic analysis was combined with proteomics of dental calculus from 33 individuals. Of the 1890 septa considered, 23 % displayed signs of periodontitis, with significantly higher rates in males. Prevalence increased with age in both sexes. Proteomic findings identified Porphyromonas gingivalis, a key periodontal pathogen, in 10 of 19 well-preserved dental calculus samples. While plaque accumulation is the main trigger for periodontitis, our findings highlight the dietary role in disease susceptibility. Carbohydrate-rich foods adhere to teeth and nourish bacteria, worsening periodontal conditions. At the same time, greater access to animal protein, particularly among emerging elites, may have contributed to inflammation. We propose that a proinflammatory diet may have been a major contributor to the proliferation of pathogenic oral microbiota.
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Pub Date : 2025-07-18DOI: 10.1016/j.jprot.2025.105492
Lanlan Li, Zhe Liu, Jinqiang Quan, Junhao Lu, Guiyan Zhao, Jun Sun
{"title":"Corrigendum to “Metabonomics analysis reveals the protective effect of nano‑selenium against heat stress of rainbow trout (Oncorhynchus mykiss)” [Journal of Proteomics 259 (2022)104545/ID: JPROT-104545]","authors":"Lanlan Li, Zhe Liu, Jinqiang Quan, Junhao Lu, Guiyan Zhao, Jun Sun","doi":"10.1016/j.jprot.2025.105492","DOIUrl":"10.1016/j.jprot.2025.105492","url":null,"abstract":"","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"320 ","pages":"Article 105492"},"PeriodicalIF":2.8,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667891","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}
Pub Date : 2025-07-18DOI: 10.1016/j.jprot.2025.105504
Carlos Estrella-Soliz , Adriana Muhlia-Almazan , Esau Bojorquez-Velazquez , Liliana Rojo-Arreola , Humberto Gonzalez-Rios , Jose A. Huerta-Ocampo
This study aims to identify and analyze the temporal variation in the activity and relative abundance of peptidases in the midgut gland of the Pacific whiteleg shrimp, Litopenaeus vannamei, during digestion. The dynamic profiles of active peptidases throughout digestion were determined by zymogram profile analysis in the midgut gland of shrimp at different feeding times: preprandial, 1 h, and 3 h postprandial. Further proteomic analysis of midgut gland extracts confirmed the identity of different-class peptidases, as well as additional isoforms not previously reported, and changes in their relative abundance. Trypsins and chymotrypsins were the predominantly active peptidases throughout the digestion process. Cathepsin D was active during the preprandial time and 3 h after ingestion, whereas a metallopeptidase showed activity at preprandial time and 1 h postprandial. Additional trypsin isoforms of varying abundances were confirmed, while chymotrypsins and cathepsin L increased significantly, peaking 3 h postprandial. The results indicate significant changes in the relative abundance of new isoforms of trypsins, cathepsins, and metallopeptidases, as well as in the previously identified peptidases involved in shrimp food protein digestion over time.
Significance
The manuscript findings revealed the peptidases' potential participation in the digestive process of the Pacific whiteleg shrimp. The integrated results from different methods confirmed the identity and active state of some of the multiple peptidase classes in the shrimp midgut gland.
Additional trypsin, cathepsin, and metallopeptidase isoforms and their abundance changes were detected before and after ingestion. These results provide additional information about the complexity and efficiency of the protein hydrolysis mechanism of L. vannamei.
{"title":"Unraveling the mechanism of peptidases participation in the protein digestion of the whiteleg shrimp (Litopenaeus vannamei)","authors":"Carlos Estrella-Soliz , Adriana Muhlia-Almazan , Esau Bojorquez-Velazquez , Liliana Rojo-Arreola , Humberto Gonzalez-Rios , Jose A. Huerta-Ocampo","doi":"10.1016/j.jprot.2025.105504","DOIUrl":"10.1016/j.jprot.2025.105504","url":null,"abstract":"<div><div>This study aims to identify and analyze the temporal variation in the activity and relative abundance of peptidases in the midgut gland of the Pacific whiteleg shrimp, <em>Litopenaeus vannamei,</em> during digestion. The dynamic profiles of active peptidases throughout digestion were determined by zymogram profile analysis in the midgut gland of shrimp at different feeding times: preprandial, 1 h, and 3 h postprandial. Further proteomic analysis of midgut gland extracts confirmed the identity of different-class peptidases, as well as additional isoforms not previously reported, and changes in their relative abundance. Trypsins and chymotrypsins were the predominantly active peptidases throughout the digestion process. Cathepsin D was active during the preprandial time and 3 h after ingestion, whereas a metallopeptidase showed activity at preprandial time and 1 h postprandial. Additional trypsin isoforms of varying abundances were confirmed, while chymotrypsins and cathepsin L increased significantly, peaking 3 h postprandial. The results indicate significant changes in the relative abundance of new isoforms of trypsins, cathepsins, and metallopeptidases, as well as in the previously identified peptidases involved in shrimp food protein digestion over time.</div></div><div><h3>Significance</h3><div>The manuscript findings revealed the peptidases' potential participation in the digestive process of the Pacific whiteleg shrimp. The integrated results from different methods confirmed the identity and active state of some of the multiple peptidase classes in the shrimp midgut gland.</div><div>Additional trypsin, cathepsin, and metallopeptidase isoforms and their abundance changes were detected before and after ingestion. These results provide additional information about the complexity and efficiency of the protein hydrolysis mechanism of <em>L. vannamei.</em></div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"320 ","pages":"Article 105504"},"PeriodicalIF":2.8,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144675046","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}
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