Molecular & Cellular Proteomics最新文献

{"title":"Optimizing NGN2 Dosage Enhances the Neuronal Enrichment of iPSC-Derived Neuronal Cultures.","authors":"Jesús Muñoz-Estrada, Andrew Mostafania, Lahiruni Halwatura, Ali Haghani, Yuming Jiang, Jesse G Meyer","doi":"10.1016/j.mcpro.2026.101604","DOIUrl":"10.1016/j.mcpro.2026.101604","url":null,"abstract":"<p><p>Proteomic analysis of highly neuron-enriched iPSC-derived cultures can provide valuable insights into the pathogenesis of neurological disease by minimizing confounding signals from non-neuronal populations. However, single-cell iPSC clones with stable NGN2 integration at the AAVS1 locus exhibit spontaneous loss of the mCherry reporter. In addition, following NGN2 induction, cultures frequently contain proliferative progenitor cells that compromise neuronal physical integrity. Here, we show that increased DNA methylation of the EF1-α promoter is associated with mCherry silencing. Importantly, reporter silencing does not affect iNeuron derivation, as evidenced by two lines of evidence. First, single-cell proteomic analysis indicated that mCherry silencing does not drive detectable proteomic divergence in undifferentiated iPSCs. Second, bulk proteomics and immunofluorescence analyses indicated that iPSC-derived neuronal cultures, whether expressing or lacking mCherry, resemble cortical glutamatergic neurons. Instead, the primary confounding factor in iNeuron generation was suboptimal neuronal conversion, which led to cell aggregates comprised of actively proliferating progenitor cells and astrocytes as the culture developed. We found that a 4-day period of NGN2 induction substantially increases neuronal maturation and the expression of neuronal-specific markers. Moreover, transient inhibition of Notch signaling is consistent with a reduction in progenitor cells. Finally, we observed that monoallelic NGN2 integration, rather than biallelic integration, preferentially increases expression of the CNS neuronal marker GPM6A over PNS markers (PRPH, POU4F1, ILS1).</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101604"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13382583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148271447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated IP-MS and Optoproteomics Reveal Dynamic Remodeling of Protein Interaction Networks During NLRP3 Inflammasome Activation.","authors":"Yuehui Zhang, Kairong Zhang, Chaozhi Jin, Limin Shang, Shanshan Wu, Mingyang Zhang, Yanzhi Yuan, Yuchen Liu, Jian Wang","doi":"10.1016/j.mcpro.2026.101595","DOIUrl":"10.1016/j.mcpro.2026.101595","url":null,"abstract":"<p><p>NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a key intracellular sensor that assembles inflammasome complexes to activate inflammatory caspases, yet the molecular composition and stimulus-dependent organization of the NLRP3 inflammasome remain incompletely defined. Here, we systematically characterized dynamic remodeling of the NLRP3 inflammasome interactome using endogenous immunoprecipitation coupled with mass spectrometry (IP-MS) targeting both NLRP3 and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) in bone marrow-derived macrophages. Quantitative interactome profiling across NLRP 3 inflammasome priming and activation states identified 1496 stimulus-associated protein interactions (1032 non-redundant), revealing extensive and stage-specific reorganization of NLRP3- and ASC-centered interaction networks. Integration of these datasets with microscopy-guided in situ protein photo-labeling (optoproteomics) of ASC specks provided spatial context to inflammasome assembly and identified inner membrane mitochondrial protein (IMMT) as an activation-associated binding partner of both NLRP3 and ASC. Functional analyses demonstrated that Immt depletion enhanced caspase-1 and gasdermin D cleavage, interleukin-1β secretion, lactate dehydrogenase release, and ASC oligomerization in response to NLRP3 agonist, indicating a negative regulatory role for IMMT in inflammasome activation. Together, this study establishes a stimulus-resolved proteomic framework for NLRP3 inflammasome interaction networks and reveals mitochondrial IMMT as a regulator of inflammasome assembly and inflammatory signaling.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101595"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13311160/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148042735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"LCAT acts as a molecular switch and regulates rooster sperm motility in vitro.","authors":"Jiwei Li, Yuzhu Cao, Juan Zhang, Yadong Tian, Guirong Sun, Yaling Gu, Xiangtao Kang, Donghua Li","doi":"10.1016/j.mcpro.2026.101598","DOIUrl":"10.1016/j.mcpro.2026.101598","url":null,"abstract":"<p><p>Sperm motility is a critical determinant of semen quality, yet the molecular mechanisms regulating motility remain unclear. In this study, we identified Yufen 1 roosters with significant differences in sperm motility among highly motile sperm samples stored under different conditions. By integrating the seminal plasma proteome and seminal plasma metabolome data, 181 proteins and 112 metabolites were identified as differentially expressed between the high motility group (HMG) and low motility group (LMG). Lecithin cholesterol acyltransferase (LCAT) was upregulated in the LMG. Targeted LCAT activation significantly reduced sperm motility and plasma membrane integrity, caused sperm plasma membrane disruption, chromatin loss, and mitochondrial swelling, and reduced the longest fertilization days and number of fertilized eggs. Mechanistically, LCAT regulates sperm motility in vitro by altering sperm membrane structure and function by modulating glycerophospholipid metabolism. These findings provide new insights into extending the window period for artificial insemination and semen preservation strategies in poultry.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101598"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13330670/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148163801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Adaptation of Cyanobacteria to Environmental Perturbations Is Achieved Through Structural Remodeling of the Proteome.","authors":"Snigdha Sarkar, Elise M Van Fossen, Xiaolu Li, Tong Zhang, Song Feng, Victoria Prozapas, Ivo Díaz Ludovico, Abdullah D Shouaib, Chelsea M Hutchinson-Bunch, Natalie C Sadler, Isaac K Attah, Wei-Jun Qian, Margaret S Cheung, Pavlo Bohutskyi, John T Melchior","doi":"10.1016/j.mcpro.2025.101443","DOIUrl":"10.1016/j.mcpro.2025.101443","url":null,"abstract":"<p><p>Dynamic environments require cyanobacteria to rapidly respond to fluctuating light conditions on timescales faster than transcription-translation processes allow, which is possible through immediate regulation of protein function via molecular and conformational adjustments. Traditional abundance-based proteomics cannot capture these rapid structural changes, creating a critical gap in understanding cellular adaptation mechanisms. We hypothesized that application of alternative structural proteomics approaches would enable identification of immediate structural remodeling across the cyanobacterial proteome triggered by environmental perturbations, potentially driving functional adaptations invisible to conventional abundance-based methods. We interrogated three complementary techniques-limited proteolysis-based mass spectrometry, thermal proteome profiling, and redox proteomics-for their capacity to unveil structural reorganization within the model cyanobacterium Synechococcus elongatus PCC 7942 during physiologically relevant light transitions. Within 30 min of increased light exposure, we detected structural changes in 753 proteins (limited proteolysis-based mass spectrometry), thermal stability shifts in 600 proteins (thermal proteome profiling), and cysteine oxidation in 1887 sites, while only 145 proteins changed in abundance. All three techniques consistently revealed coordinated remodeling of photosynthetic machinery, ribosomal complexes, and carbon metabolism, exemplified by cytochrome f stabilization modulating electron transport efficiency. Remarkably, <10% of proteins overlapped between methods, demonstrating that each technique captures distinct molecular dimensions of environmental adaptation. This structural proteomics framework demonstrates how alternative techniques can reveal hidden facets of proteome dynamics underlying cellular processes, offering new methodological approaches for understanding environmental responses and informing biotechnological applications.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101443"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13424402/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145452375","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Limited Impact of Column Chemistry and Length on Proteome Coverage Under High-Speed DIA.","authors":"Alicia-Sophie Schebesta, Kathrin Korff, Ericka C M Itang, Vincent Albrecht, Philipp E Geyer, Johannes B Mueller-Reif","doi":"10.1016/j.mcpro.2026.101609","DOIUrl":"10.1016/j.mcpro.2026.101609","url":null,"abstract":"<p><p>The evolution of mass spectrometry (MS)-based proteomics has been driven by continuous technological advances in sample preparation, liquid-phase separations, instrumentation, and data acquisition. Chromatographic performance has been recognized as a contributing factor to identification depth, particularly on earlier-generation MS platforms. Recent advances in MS sampling speed and sensitivity now raise the question of how strongly chromatographic quality continues to determine overall proteome coverage. We investigate how column chemistry and length influence proteome coverage and chromatographic selectivity under modern data-independent acquisition conditions, and whether traditional optimization priorities still apply. Spanning a matrix of experiments with five distinct stationary phases, including C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluate protein identification performance using data-independent acquisition on the Orbitrap Astral mass spectrometer. Despite differences in stationary-phase chemistry and column length, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor- and approximately 9000 protein group identifications, regardless of column length variations. While retention fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in proteome coverage under high-speed acquisition conditions at 200 Hz. Within the range of modern sub-2 μm reversed-phase materials tested, identification depth showed limited dependence on column chemistry and length, suggesting that for state-of-the-art stationary phases, method development priorities may increasingly favor operational robustness, throughput, and reproducibility over traditional separation optimization.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101609"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13400878/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148302077","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"π-SeqOmics: A Sequential Workflow for Genomic, Transcriptomic, Proteomic, and Phosphoproteomic Profiling From Biopsy-Scale Samples.","authors":"Shuyi Feng, Xuehui Deng, Ying Xu, Baoyi Qin, Chuanxi Huang, Qingjing Chen, Fuchu He, Dongxue Wang","doi":"10.1016/j.mcpro.2026.101596","DOIUrl":"10.1016/j.mcpro.2026.101596","url":null,"abstract":"<p><p>Comprehensive multiomics profiling from a single limited specimen remains challenging, as current extraction methods are difficult to standardize and often compromise balanced recovery of nucleic acids and proteins. Here, we developed π-SeqOmics, a phenol-free workflow established through systematic benchmarking for the sequential isolation of DNA, RNA, and proteins from a single specimen, enabling integrated genomic, transcriptomic, proteomic, and phosphoproteomic analyses. Compared with conventional extraction and in-solution digestion workflows, π-SeqOmics achieves comparable proteomic and phosphoproteomic depth and reproducibility while preserving high-quality DNA and RNA for sequencing. The workflow performed robustly across a wide input range (5 × 10⁶ - 1 × 10⁵ cells) and multiple mouse tissues, consistently identifying over 8000 proteins with strong quantitative reproducibility (r > 0.95). Together, these results establish π-SeqOmics as a standardized, practical, and cost-effective multiomics sample preparation workflow for limited-input and biopsy-scale specimens, providing a robust foundation for systems biology and translational research.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101596"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13312471/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148055841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DORSSAA: Drug-Target interactOmics Resource Based on Stability/Solubility Alteration Assay.","authors":"Ehsan Zangene, Elham Gholizadeh, Veit Schwämmle, Amir Ata Saei, Mathias Wilhelm, Lukas Käll, Mohieddin Jafari","doi":"10.1016/j.mcpro.2026.101603","DOIUrl":"10.1016/j.mcpro.2026.101603","url":null,"abstract":"<p><p>Advancements in high-throughput techniques such as Thermal Proteome Profiling and the high-throughput Proteome Integral Solubility Alteration assay have revolutionized our understanding of drug-protein interactions. Despite these innovations, the absence of an integrative platform for cross-study analysis of stability and solubility alteration data represents a significant bottleneck. To address this gap, we introduce Drug-target interactOmics Resource based on Stability/Solubility Alteration Assay (DORSSAA), an interactive and expandable web-based platform for the systematic analysis and visualization of proteome stability and solubility alteration assay datasets. Currently, DORSSAA features 1,135,985 records spanning 38 cell lines and organisms, 135 compounds, and 40,742 protein targets. Through its user-friendly interface, the resource supports comparative drug-protein interaction analysis and facilitates the discovery of actionable therapeutic targets. Through two case studies, methotrexate target profiling in A549 cells and combinatorial-therapy drug-target interactions in leukemia cell lines, we demonstrate DORSSAA's utility for identifying protein-drug interactions across diverse experimental contexts. This resource empowers researchers to accelerate drug discovery and enhance our understanding of protein behavior. Compared with data repositories and interaction databases, DORSSAA provides direct protein-level evidence of mechanisms of action with strict statistical control for each study. This enables more reliable identification of drug targets, off-target effects, and potential drug combinations.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101603"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13380709/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148271408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Both L-Lactyl and D-Lactyl Enantiomers Modify Histones in Mouse Testis.","authors":"Julie Manessier, Hassan Hijazi, Lisa Vizzini, Sabine Brugière, Marie Courçon, Christophe Masselon, Alberto de la Iglesia, Julie Cocquet, Delphine Pflieger","doi":"10.1016/j.mcpro.2026.101601","DOIUrl":"10.1016/j.mcpro.2026.101601","url":null,"abstract":"<p><p>Dynamic histone posttranslational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here, we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L-over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and postmeiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25 to 35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101601"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13380202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148239492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Extracellular Vesicles From Glioblastoma Cells Reflect 2D vs. 3D Culture Adaptation and Resistance to Temozolomide.","authors":"Getulio Pereira de Oliveira, Alan Zimmerman, Cintia C Palu, Vivian Tran, Gabrielle Bogut, Stephanie Chidester, John Tigges, Rafael A Vega, Elena Aikawa, Jennifer Jones, Ionita C Ghiran, Alexander R Ivanov","doi":"10.1016/j.mcpro.2026.101610","DOIUrl":"10.1016/j.mcpro.2026.101610","url":null,"abstract":"<p><p>Glioblastoma (GBM) is an aggressive brain tumor marked by extensive heterogeneity, resistance to therapy, and dismal prognosis. Extracellular vesicles (EVs) have emerged as key players in GBM biology, mediating intercellular communication and therapy adaptation. However, the exact functions and molecular impact of EVs in GBM remain incompletely understood. In this study, we performed a comparative proteomic analysis of U87MG GBM cells grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroids following temozolomide (TMZ) treatment, alongside characterization of EVs derived from both culture systems. 3D-spheroids secreted more EVs of smaller size and exhibited a more TMZ-resistant, stem-like proteome under TMZ-induced genotoxic stress. In contrast, 2D cell cultures demonstrated greater proteome remodeling, with EVs enriched in protein families involved in DNA repair, oxidative stress adaptation, and methylation processes. Notably, several methyltransferases were decreased intracellularly but selectively retained in EVs, suggesting active sorting to influence the tumor microenvironment or modulate epigenetic states in recipient cells. EVs also carried adhesion molecules and signaling proteins linked to migration, invasion, and Wnt pathway activation, as well as metabolic enzymes connecting serine metabolism and redox control to TMZ resistance. Mapping EV and cellular proteomes onto The Cancer Genome Atlas (TCGA) dataset identified prognostic protein families associated with either poor or favorable patient outcomes. Our data demonstrate that EV cargo composition mirrors TMZ-induced phenotypic adaptation and reveals molecular mechanisms underlying therapeutic resistance. These EV-associated signatures may serve as clinically actionable biomarkers for patient stratification and offer potential targets to overcome chemoresistance in GBM.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101610"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13393710/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148308764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"dia-PASEF Enables Rapid Profiling of the Human Secretome for Deeper Insights Into Cellular Dynamics and Inflammatory Mechanisms.","authors":"Chloe L Tayler, Serena Bateman, Charlie Haslam, Leonie Müller, Kerena Norris, Evan Rosa-Roseberry, Stan Martens, Jonathan Yu, Eleanor Dickinson, Lee Booty, Rebecca Beveridge, Nicholas J W Rattray, Rachel Peltier-Heap","doi":"10.1016/j.mcpro.2026.101597","DOIUrl":"10.1016/j.mcpro.2026.101597","url":null,"abstract":"<p><p>Protein secretion is a fundamental mechanism for cellular coordination and signaling, with its dysregulation leading to widespread physiological dysfunction and disease. Immunoassay formats that utilize secondary antibody readouts are the current gold standard for measuring secreted proteins, offering high specificity and sensitivity, but relying on predefined protein panels that constrain the discovery of novel biology. We present a scalable mass spectrometry-based workflow that combines data-independent acquisition with ion mobility and parallel fragmentation to deliver rapid, global profiling of the secretome. Using a translationally relevant human iPSC-derived macrophage model, our approach robustly identified over 900 protein groups in under 15 min of acquisition time, delivering exceptional reproducibility across a large sample set. We applied this approach to profile proinflammatory phenotypes, confirming robust identification of key cytokines and chemokines whilst revealing non-canonical immune responses absent from both targeted panels and the intracellular proteome. In particular, we identified a unique cholesterol efflux signature, marked by the secretion of APOA1 and PON1, in response to Mycobacterium tuberculosis, consistent with the metabolic reprogramming that takes place during infection. Furthermore, temporal profiling of macrophage responses to lipopolysaccharide over 24 h resolved dynamic secretion trajectories that distinguish between acute and chronic inflammatory states. The extended time period facilitated the observation of distinct cytokine-dependent secretion phenotypes, with early secretion of TNF and IL-6 initiating downstream signaling cascades that resulted in the delayed secretion of chemokines such as CXCL10 and CCL8. Collectively, these findings establish a robust, scalable platform for global characterization of secretory networks. Beyond macrophage biology, this workflow offers broad utility for biomarker discovery, mechanistic studies of disease progression and evaluation of new therapeutic interventions, providing a powerful tool for advancing precision medicine.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101597"},"PeriodicalIF":6.3,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13314884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148055880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}