Journal of proteomics最新文献

{"title":"Early regulatory networks driving somatic embryogenesis in Saccharum spp. L. revealed by time-resolved proteomics","authors":"Roberta Pena da Paschoa ,&nbsp;Lucas Rodrigues Xavier ,&nbsp;Caio Cezar Guedes Corrêa ,&nbsp;Karina da Silva Vieira ,&nbsp;Daniel Dastan Rezabala Pacheco ,&nbsp;Lucas do Espirito Santo Gomes ,&nbsp;Carlos Eduardo Assis da Silva ,&nbsp;Laura Eliza de Oliveira Alves ,&nbsp;Vitor Batista Pinto ,&nbsp;Claudete Santa-Catarina ,&nbsp;Vanildo Silveira","doi":"10.1016/j.jprot.2026.105602","DOIUrl":"10.1016/j.jprot.2026.105602","url":null,"abstract":"<div><div>The induction of somatic embryogenesis is controlled by various genes and proteins involved in hormonal pathways and stress responses, which act as key regulators of in vitro cellular reprogramming. In this study, we employed a temporal proteomic approach to investigate the underlying molecular mechanisms governing sugarcane (<em>Saccharum</em> spp.) embryogenic callus formation in response to 2,4-dichlorophenoxyacetic acid (2,4-D) during induction. Proteomic profiling revealed 996 differentially accumulated proteins (DAPs) across at least one pairwise comparison among time points (0, 7, 14 and 21 days) during callus induction. These DAPs were classified into different clusters on the basis of their accumulation profile. Proteins involved in embryogenesis, histone epigenetic regulation, hormone responses and protein post-translational modification accumulate during callus induction. The predicted interactions between the TOPLESS protein and auxin response proteins (SKP1, CUL1 and CAND1) are associated with increased accumulation of the histone deacetylase HDT2 protein, a regulator of chromatin condensation, during embryogenic callus initiation. Moreover, proteomic analysis revealed a temporal reduction in methylation cycle enzymes during callus induction, whereas global DNA methylation showed only a slight, non-significant increase, suggesting that additional regulatory layers are present. The identified protein dynamics provide valuable targets for refining somatic embryogenesis protocols and advancing their biotechnological applications in sugarcane.</div></div><div><h3>Significance</h3><div>Genetic engineering and plant cloning usually involve the induction of embryogenic competence using 2,4-dichlorophenoxyacetic acid (2,4-D). This study presents protein-protein interaction (PPI) networks regulated during the induction of sugarcane callus using 2,4-D, in addition to the morphological aspects of the explant during the process. Proteomic analysis of time series shows the regulation of protein kinases and transcriptional regulators TOPLESS, CUL1, SKP1, CAND1, and ARGONAUTE kinases, revealing mechanisms of activation of induction and multiplication of embryogenic callus. Furthermore, the possible interaction between GH3.8 and SnRK/SAPK kinases suggests a link between hormonal responses.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"326 ","pages":"Article 105602"},"PeriodicalIF":2.8,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986725","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":"Emphasizing the importance of interactions and networks in proteomics.","authors":"Jean Armengaud","doi":"10.1016/j.jprot.2026.105599","DOIUrl":"https://doi.org/10.1016/j.jprot.2026.105599","url":null,"abstract":"","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":" ","pages":"105599"},"PeriodicalIF":2.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145985097","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 signatures in triple-negative breast cancer","authors":"Sohit Kashyap ,&nbsp;Vishal Patidar ,&nbsp;Anil Kumar ,&nbsp;Prachi Sahu ,&nbsp;Monisha Dhiman ,&nbsp;Pardeep Garg ,&nbsp;Aklank Jain ,&nbsp;Anjana Munshi","doi":"10.1016/j.jprot.2026.105598","DOIUrl":"10.1016/j.jprot.2026.105598","url":null,"abstract":"&lt;div&gt;&lt;div&gt;An aggressive and heterogeneous malignancy, referred to as triple-negative breast cancer, is characterised by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Treatment options remain limited, relying primarily on chemotherapy due to the lack of well-defined therapeutic targets, which is linked with poor prognosis and high recurrence rates. Proteomics and other high-throughput technologies have significantly advanced TNBC research by enabling the identification of protein-based biomarkers with potential applications in diagnosis, prognosis, and treatment. Through protein biomarkers that affect immune checkpoints, cell-surface glycoproteins, and regulators of tumor microenvironment interactions, key protein signatures from tumor tissue, serum, and exosomal proteomics have been found to have the potential to predict chemotherapy response and disease progression. To develop new therapeutic approaches, these biomarkers are being investigated. Combining proteomics with other omics technologies, such as transcriptomics and genomics, enables the development of precision medicine approaches and provides deeper insights into the pathophysiology of TNBC. Clinically validated and newly developed protein biomarkers for diagnosis, prognosis, and treatment interventions are described in this review. The molecular mechanistic aspects have also been discussed. These biomarkers have the potential to aid in the classification, risk stratification, and development of personalized treatment approaches for TNBC.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Significance statement&lt;/h3&gt;&lt;div&gt;Triple-negative breast cancer (TNBC) remains a highly aggressive and heterogeneous form of breast cancer, with very few treatment strategies. This review synthesizes past discoveries, current clinical applications, and future opportunities of proteomics in TNBC, making it highly relevant to the theme of this Special Issue on “Past, Present and Future of Proteomics.” By consolidating evidence from human and other preclinical studies, it highlights how proteomic signatures have already transformed our understanding of TNBC biology and subtype classification, while also outlining their growing impact as diagnostic, prognostic, and therapeutic markers.&lt;/div&gt;&lt;div&gt;Importantly, the review emphasizes the translational shift enabled by next-generation proteomic technologies to redefine precision medicine for TNBC. It showcases how proteomics can facilitate personalized medicine, drug repurposing, and rational combination therapies, and describes novel avenues such as single-cell proteomics and integrative immunoproteogenomics that are driving the field forward.&lt;/div&gt;&lt;div&gt;Thus, this work not only consolidates what has been achieved but also provides perspectives on emerging technologies and innovative applications that could revolutionize biomarker discovery and clinical management of TNBC. It highlights proteomics as a critical pillar in shaping the futu","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105598"},"PeriodicalIF":2.8,"publicationDate":"2026-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145927329","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":"Combining information on degradomics and gene expression data in prospecting metastatic melanoma proteolytic signatures","authors":"Murilo Salardani ,&nbsp;Alison F.A. Chaves ,&nbsp;Leonardo Cardili ,&nbsp;Miyuki Uno ,&nbsp;Solange M.T. Serrano ,&nbsp;Roger Chammas ,&nbsp;André Zelanis","doi":"10.1016/j.jprot.2025.105597","DOIUrl":"10.1016/j.jprot.2025.105597","url":null,"abstract":"<div><div>Melanoma is an aggressive skin cancer with a high metastatic potential, influenced by both genetic and environmental factors. Proteases play a key role in shaping the tumor microenvironment and enabling transformed cells to actively colonize distant sites (metastasis). We performed proteomic mapping of protease cleavage sites in formalin-fixed paraffin-embedded tissue samples and profiled potentially active proteases in samples from melanoma patients with distinct prognostic outcomes. Although protein abundance alone did not indicate potential markers of disease progression, the observed cleaved fragments may serve for monitoring potentially active proteases in patient samples in targeted proteomics analysis. The findings provide valuable insights into melanoma biology and potential therapeutic prospects.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105597"},"PeriodicalIF":2.8,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145892564","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 comparison of human brain tissue preservation methods","authors":"Laura Plantera ,&nbsp;Anna Didio ,&nbsp;Uta Ceglarek ,&nbsp;Ingo Bechmann","doi":"10.1016/j.jprot.2025.105589","DOIUrl":"10.1016/j.jprot.2025.105589","url":null,"abstract":"<div><div>This study investigated the impact of tissue preservation methods on protein profiles analyzed by reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using data-independent acquisition (DIA). Proteomic profiles from formalin-fixed, formalin-fixed and paraffin-embedded (FFPE), and fresh-frozen human brain tissues (cortex and hippocampus, <em>n</em> = 6) were compared, including an FFPE-specific protein extraction kit (<em>n</em> = 4).</div><div>Formalin-fixed samples more closely resembled fresh-frozen profiles than FFPE or FFPE-Kit samples, while still showing high correlation and overlap with FFPE tissues in principal component analyses. A core set of 1753 proteins was consistently detected across all sample preparation methods. A total of 35 proteins were identified exclusively in fresh-frozen samples, but without functional enrichment. Quantitative comparisons to the proteome of fresh-frozen tissue revealed an underrepresentation of cellular processes, energy metabolism, signaling, and transport related to protein properties such as length, location, and hydrophobicity. In contrast, neuronal development and phagosome-related pathways were overrepresented in fixed tissues.</div><div>In a pilot study comparing low (Braak 0-II, <em>n</em> = 4) and high (Braak IV-VI, n = 4) Alzheimer's disease (AD) stages using formalin-fixed samples, we identified 12 potential protein biomarkers, primarily nucleosomal proteins and carboxypeptidase M (CPM).</div><div>These findings suggest that formalin-fixed brain tissue provides reliable proteomic information, making it a valuable resource for neurodegenerative disease research.</div></div><div><h3>Significance</h3><div>Proteomics offers enormous potential for investigating the molecular regulation of the human brain. Valuable tissue samples are often preserved in formalin or additionally with paraffin for later analysis. The potential value of these preserved samples for proteomic analysis has already been recognized. However, tissue preservation poses a challenge for proteome analysis. Consequently, several studies have compared different protein extraction protocols for fixed samples. In addition, studies have been published comparing protein extraction from FFPE samples with fresh-frozen samples. To our knowledge, this is the first study to compare protein extraction across all three tissue preservation methods with subsequent functional analysis using samples obtained from the same donors, thereby eliminating inter-donor variability and enabling a direct comparison of preservation effects. This study validates a protein extraction protocol from formalin-fixed samples, laying the groundwork for future research into potential biomarkers in formalin-fixed samples.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105589"},"PeriodicalIF":2.8,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145882649","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":"Assessing systemic effects of Bothrops jararaca venom in the lungs in a mouse model by label-free proteomics using DDA and DIA","authors":"Ana Luiza T. Silva ,&nbsp;Bianca C.S.C. de Barros ,&nbsp;Joanderson P.C. da Silva ,&nbsp;Isabel S. Carvalho ,&nbsp;Marcelo L. Santoro ,&nbsp;Alison F.A. Chaves ,&nbsp;Solange M.T. Serrano","doi":"10.1016/j.jprot.2025.105588","DOIUrl":"10.1016/j.jprot.2025.105588","url":null,"abstract":"<div><div>Snakebite envenomation is a critical yet underexplored public health issue, particularly in tropical and subtropical regions. <em>Bothrops jararaca</em> venom induces severe local and systemic effects, including pulmonary injury, however, the molecular mechanisms underlying lung tissue damage remain poorly understood. This study employed label- free quantitative proteomics to map protein alterations in the lung tissue in a mouse model of envenomation. Using Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) approaches combined with different sample preparation methods, we provide a comprehensive proteomic profile of venom-induced pulmonary damage. Our findings reveal significant changes in proteins involved in inflammatory responses, extracellular matrix remodeling, oxidative stress, and blood coagulation. Comparative analyses highlight the superior performance of DIA over DDA, with DIA offering deeper proteome coverage, enhanced detection of low-abundance proteins, and improved resolution of venom-induced alterations. This benchmark study underscores the potential of DIA as a robust tool for elucidating complex, systemic, mammalian molecular responses to animal toxins. By bridging the gap between proteomic methodologies and pathophysiological insights, our findings contribute to a deeper understanding of viperid snake venom-induced lung injury and give insights for improved clinical management strategies.</div></div><div><h3>Significance</h3><div><em>Bothrops</em> snakebites remain a major neglected health problem, causing severe local and systemic complications. While the impact of viperid venoms on muscle and kidney tissues is well documented, the effects on the lungs — an organ critically involved in systemic envenomation outcomes — remain poorly understood. Our study provides the first comprehensive proteomic characterization of lung responses to <em>B. jararaca</em> venom in a murine model, revealing alterations in pathways related to inflammation, extracellular matrix remodeling, oxidative stress, and coagulation. These molecular insights fill an important knowledge gap by showing that proteomic disturbances occur even in the absence of overt lung pathology, highlighting the lungs as a key systemic target of envenomation. Moreover, by demonstrating that antivenom administration mitigates many of these changes, our findings underscore both the therapeutic efficacy of antivenom and the need to better understand its broader systemic footprint. This work advances both toxinology and proteomics by linking molecular-level disturbances to clinically relevant systemic outcomes.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105588"},"PeriodicalIF":2.8,"publicationDate":"2025-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145850154","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":"Salivary proteomics and metabolic responses to resistance training with and without blood flow restriction in young adults","authors":"Gustavo Zanete Alencar ,&nbsp;Dalton Muller Pessôa Filho ,&nbsp;Karina Oliveira Santos ,&nbsp;Anderson Geremias Macedo ,&nbsp;Henrique Basso Vitti ,&nbsp;Guilherme Neves Gasparino ,&nbsp;Larissa Tercília Grizzo Thomassian ,&nbsp;Murilo Henrique Faria ,&nbsp;Ana Carolina Magalhães","doi":"10.1016/j.jprot.2025.105587","DOIUrl":"10.1016/j.jprot.2025.105587","url":null,"abstract":"<div><div>Blood flow restriction (BFR) has been applied as alternative strategy to reproduce the effects of conventional high-load intensity training (HI) while training with low-loads (LI). Therefore, the aim of this research was to evaluate the salivary proteomic and metabolic responses to different resistance training in young adults.Ten participants were selected and underwent to training with LI (30 %1RM – one repetition maximum) plus BFR vs. HI (70 %1RM) without BFR, at 48 h-interval. Stimulated saliva was collected before and immediately after the sessions, while breath-by-breath oxygen uptake (VO₂) was measured during and after each session. Arterial blood samples for lactate concentration measurement (in EqO_2[La]) were taken at 1^st minute of resting between each exercise. For HI, there was an increase in two actin cytoplasmic isoforms and two immunoglobulin isoforms and a decrease of six hemoglobin isoforms. For LI-BFR, there was an increase in two hemoglobin isoforms, and the same immunoglobulin isoforms (<em>t</em>-test, <em>p</em> &lt; 0.05). No differences were significant between HI and LI + BRF training regarding the total energy demand (in mlO₂), absolute oxygen values (mlO₂) for oxidative response (VO_2ON), glycolytic (EqO_2[La]), and oxygen debt (VO_2OFF) (<em>p</em> &gt; 0.05). Both HI and LI + BFR protocols modulated immune system activity and exhibited divergent hemoglobin patterns.</div></div><div><h3>Significance</h3><div>The current study, which identified protein from saliva samples, a non-invasive method, and analyzed physiological markers, enabled the comparison of different resistance exercise protocols. Although no significant differences were observed in the metabolic responses to each protocol, which highlights the potential of LI + BRF to reproduce a high-intensity training stimulus, the changes in salivary protein profiles indicate specific functional adaptations that may become evident over time.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"325 ","pages":"Article 105587"},"PeriodicalIF":2.8,"publicationDate":"2025-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145843898","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":"MALDI Deamidation Score (MDS): A fast and flexible method for assessing deamidation in ZooMS data and its application to the Denisova Cave bone assemblage","authors":"Fei Yang ,&nbsp;Ismael Rodríguez Palomo ,&nbsp;Bharath Anila Bhuvanendran Nair ,&nbsp;Samantha Brown","doi":"10.1016/j.jprot.2025.105577","DOIUrl":"10.1016/j.jprot.2025.105577","url":null,"abstract":"&lt;div&gt;&lt;div&gt;Estimating deamidation from ZooMS spectra has frequently been achieved using the q2e method due to its high-throughput capacity and ease of use. Despite this, q2e only works with spectral data in txt format, operates slowly, and employs a genetic algorithm for fitting, which is stochastic and less interpretable. Furthermore, q2e only estimates deamidation at the peptide level and does not generate a sample-wide summary. The introduction of the Parchment Glutamine Index (PQI) presented an alternative method. Initially designed for a large ZooMS dataset of parchment, it utilises weighted least squares and a linear mixed-effects model (LME) to generate a peptide deamidation estimation and a sample level index, respectively. To address the limitations of q2e and expand the applicability of PQI to a wider range of archaeological tissues and MALDI-TOF-derived data (such as ZooMS data on bone collagen), we developed the MALDI Deamidation Score (MDS), an iteration of the PQI method optimised for handling large-scale datasets. MDS is more streamlined for analysing multi-species data with customisable peptide lists, offering dramatically reduced processing time. Using the published Denisova Cave ZooMS assemblage, we demonstrate that different peptides exhibit varying deamidation patterns over time, making the use of a single peptide to represent overall deamidation potentially biased. Such information is invaluable for investigating key questions such as protein preservation and site formation processes.&lt;/div&gt;&lt;/div&gt;&lt;div&gt;&lt;h3&gt;Significance&lt;/h3&gt;&lt;div&gt;We introduce the MALDI Deamidation Score (MDS), a new model that estimates deamidation at both peptide and sample levels from MALDI-derived datasets. MDS streamlines multi-species analyses with customisable peptide lists and markedly reduces processing time, enabling robust and large-scale deamidation estimation. Before MDS, deamidation in ZooMS has often been inferred from a single peptide common to Eurasian terrestrial mammals. Applied to the published Denisova Cave ZooMS assemblage, MDS reveals peptide-specific temporal deamidation patterns, showing that single-peptide proxies can be biased as peptides differ in deamidation rates and may be subject to different taphonomic processes. These results have broad relevance for studies of protein preservation and site-formation processes related to molecular taphonomy across archaeological and paleontological contexts. Looking ahead, as MDS allows a customisable peptide list, it has the potential to extend deamidation analysis of ZooMS data beyond terrestrial mammals to marine mammals and non-mammalian vertebrates (e.g., birds), and to different proteinaceous material such as keratin. It also has the potential to be compatible with different instruments used in peptide mass fingerprinting, such as MALDI-TOF, MALDI-FTICR, and TIMS-TOF. We believe that MDS will contribute greatly to our understanding of deamidation on different peptide sequences under various e","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"324 ","pages":"Article 105577"},"PeriodicalIF":2.8,"publicationDate":"2025-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145661494","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":"Sulfur metabolism regulates endoplasmic reticulum stress survival through the interaction between cystathionine beta-synthase and Sec14 protein","authors":"Elias Nieto-Zaragoza ,&nbsp;Emilio Espinoza-Simón ,&nbsp;Damián Ramirez-Robles ,&nbsp;Emmanuel Ríos-Castro ,&nbsp;Ramiro Garza-Domínguez ,&nbsp;Francisco Torres-Quiroz","doi":"10.1016/j.jprot.2025.105573","DOIUrl":"10.1016/j.jprot.2025.105573","url":null,"abstract":"<div><div>The gasotransmitter hydrogen sulfide (H₂S) is implicated in a myriad of biological processes, including disease causing alterations. Besides acting as an antioxidant molecule, H₂S reacts with thiol groups from cysteine residues found in proteins. This post-translational modification (PTM), called S-persulfidation, plays an essential role during endoplasmic reticulum (ER) stress. Here we demonstrated that yeast cystathionine beta-synthase (Cys4), is the main responsible for ER H₂S-mediated homeostasis. We also found that during ER stress, Cys4 interacts with specific proteins that we identified with at least one cysteine residue modified by hydrogen sulfide. Finally, we focused on one interactor with reported tunicamycin sensitivity, the phospholipid transfer protein Sec14. In addition to Cys4 immunoprecipitation, we confirmed this interaction by pulling down Sec14 interactors. Deleting <em>CYS4</em> generated the accumulation of lipid droplets inside the cytosol, similar to Sec14. For the first time, we described a set of protein-protein interactions of Cys4 during ER stress with cysteines susceptible to S-persulfidation, leaving an open question about the role and regulation of the rest of the interactors.</div></div><div><h3>Significance</h3><div>In this study, we identified several interactor partners of yeast cystathionine beta synthase and analyzed cysteine residues with cysteine S-persulfidations. Although yeast have multiple H₂S producing enzymes, Cys4 seems to play a predominant role during ER stress. Temporal and spatial generation of H₂S is becoming significant in the field, and our findings contribute to that understanding. Besides, Cys4/Sec14 complex found here, seven different Cys4 complexes that may be regulated by H₂S were also reported. Because the biogenesis of this PMT is still controversial, findings like these support the hypothesis of interaction mediated biogenesis. H₂S producing enzymes specificity would help control their signaling across the cell. Finally, all Cys4 interactors with modified cysteines, including Sec14, are excellent targets to study how this PTM regulates enzyme functioning. Structural and functional assays with cysteine mutants in these proteins will shed light on new regulatory mechanisms.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"324 ","pages":"Article 105573"},"PeriodicalIF":2.8,"publicationDate":"2025-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145653287","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":"Opt-TMT: An optimized and scaled-down TMT labeling strategy for limited sample","authors":"Michele Martins,&nbsp;Fábio César Sousa Nogueira,&nbsp;Magno Junqueira,&nbsp;Guillaume Nugue","doi":"10.1016/j.jprot.2025.105576","DOIUrl":"10.1016/j.jprot.2025.105576","url":null,"abstract":"<div><div>Mass spectrometry-based proteomics has evolved and currently requires minimal sample quantities. However, manufacturers' isobaric labeling protocols, such as Tandem Mass Tag (TMT), are still designed for larger sample amounts, leading to significant costs and limiting research possibilities. Here, we present an optimized isobaric labeling protocol (Opt-TMT) that maintains high labeling efficiency while substantially reducing reagent consumption and sample requirements. We achieved consistent labeling efficiency even with peptide quantities as low as 6–15 μg per channel by adjusting reaction conditions, including volume reduction and increased peptide concentration. Importantly, our protocol reduces reagent costs by up to 90 % compared to the commercial protocol, while maintaining labeling efficiency above 99 %. This cost-effective approach addresses key challenges in proteomic research, especially for studies involving limited biological material or post-translational modification analyses. The Opt-TMT method provides a practical solution for researchers seeking to maximize the utility of isobaric labeling while minimizing resource expenditure, without compromising analytical quality.</div></div><div><h3>Significance</h3><div>The Opt-TMT protocol provides a major advancement for proteomics by making isobaric labeling both cost-effective and scalable to very limited sample amounts. By reducing reagent costs up to 90 % without compromising labeling efficiency (&gt;99 %), this approach enables high-quality quantitative proteomics for studies where material is scarce, such as patient-derived samples, biopsies, or post-translational modification analyses. Importantly, Opt-TMT broadens access to TMT-based workflows for laboratories with limited resources, while preserving analytical robustness. This work contributes a practical and impactful methodological improvement that directly benefits both fundamental and applied proteomics research.</div></div>","PeriodicalId":16891,"journal":{"name":"Journal of proteomics","volume":"324 ","pages":"Article 105576"},"PeriodicalIF":2.8,"publicationDate":"2025-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145635030","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}