Molecular & Cellular Proteomics最新文献

{"title":"Micro- and Macro-Heterogeneity of N-Glycoproteome Remodeling in Endoplasmic Reticulum Stress.","authors":"Alexander Black, Tiffany Ngo, Boomathi Pandi, Andrew R Mongin, Dominic C M Ng, Edward Lau, Maggie P Y Lam","doi":"10.1016/j.mcpro.2026.101653","DOIUrl":"10.1016/j.mcpro.2026.101653","url":null,"abstract":"<p><p>N-glycosylation plays essential roles in the folding, trafficking, and maturation of proteins in the secretory pathways, but how individual protein- and site-specific glycosylation rewires under endoplasmic reticulum (ER) stress is unknown. Particularly, intact glycopeptide data that retain the connectivity between glycosylation sites and the attached glycans are needed to reveal the micro- and macro-heterogeneity of N-glycosylation sites and their permutations in stressed cells. Here, we developed and optimized a magnetic polyethylenimine boronic acid-containing scaffold (mPBA) enrichment workflow to achieve sensitive and broad enrichment of intact glycopeptides for mass spectrometry analysis, requiring only 0.1 to 0.5 mg total peptide input. With this method, we performed a large intact glycopeptide comparative study, systematically analyzing 13,759 unique protein-, site-, and glycoform combinations, termed glycopeptidoforms, in normal and stressed human cells. The data reveal a dynamic rewiring of N-glycosylation involving hundreds of proteins with complex protein-, site-, and glycan-specific granularity. The magnitude of differential glycosylation far exceeds that of protein expression changes. Individual glycoform reconfigurations can be observed that indicate likely disruptions within specific steps in protein maturation and trafficking. Mannose trimming emerges as a shared disruption across multiple proteins, suggesting a processing bottleneck of the ER stress glycoproteome. Together, these results reveal molecular details into the remodeling of protein secretory pathways upon ER stress and highlight the utility of mPBA for sensitive N-glycoproteomics studies. The data can be visualized on https://glycoproteome.info.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101653"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13639171/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148874607","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":"Systematic Workflow Optimization for Ultra-sensitive Targeted Immunopeptidomics.","authors":"Jonas D Förster, Jonas P Becker, Nika Vučković, Angelika B Riemer","doi":"10.1016/j.mcpro.2026.101634","DOIUrl":"10.1016/j.mcpro.2026.101634","url":null,"abstract":"<p><p>Epitope detection sensitivity remains a primary bottleneck in mass spectrometry (MS)-based immunopeptidomics, as conventional discovery-based workflows such as data-dependent (DDA) and data-independent acquisition (DIA) frequently lack the sensitivity required to detect ultra-low abundant targets. While these untargeted methods are powerful for mapping the general immunopeptidome, the stochastic nature of precursor selection and the presence of complex, chimeric spectra mean that rare species, such as viral or mutation-derived neoepitopes, often remain undetected. In this study, we present optiPRM+, an ultra-sensitive targeted-first workflow for the Orbitrap Exploris 480 platform that integrates systematically optimized targeted acquisition with untargeted DIA contextualization to bridge this sensitivity gap. Our approach centers on the empirical characterization of target peptides using direct infusion-MS to determine optimal fragmentation conditions and inclusion list-driven data-dependent acquisition (iDDA). To maximize signal-to-noise ratios for these trace-level targets, we employed ultra-high MS2 resolutions (up to 480,000), ion injection times up to 1000 ms, and narrow precursor isolation windows. Additionally, we discovered that precursors with a charge state exceeding their basic amino acid count require unusually low energies for optimal fragmentation, which is especially relevant for the non-tryptic peptides characteristic of the immunopeptidome. We applied the optiPRM+ workflow to the challenging biological case of Human Papillomavirus type 16 (HPV16), a virus known to suppress antigen presentation pathways. This optimized strategy enabled the confident identification and validation of the human leukocyte antigen (HLA)-A∗02:01-restricted epitope TIHDIILECV and, to our knowledge, the first MS-based detection of two novel viral targets: ISEYRHYCY (HLA-A∗01:01) and CVYCKQQLLR (HLA-A∗11:01). Subsequent global immunopeptidome analysis via DIA confirmed that these ultra-low abundance peptides were not detectable through untargeted methods despite being clearly validated by our targeted approach. By successfully detecting these viral peptides, we demonstrate that a systematically optimized targeted-first approach can uncover biologically relevant epitopes that remain invisible to conventional discovery-based workflows.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101634"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13545375/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148701841","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":"Sugar-Coded Signals: Glycan-Based Biomarkers in Rheumatoid Arthritis.","authors":"Michal Hires, Stefania Hroncekova, Viera Dujnič, Veronika Solovicova, Veronika Pinkova Gajdosova, Eduard Jane, Lenka Lorencova, Tomas Bertok, Alica Vikartovska, Anna Datkova, Peter Kasak, Radek Kucera, Jan Tkac","doi":"10.1016/j.mcpro.2026.101633","DOIUrl":"10.1016/j.mcpro.2026.101633","url":null,"abstract":"<p><p>This review paper summarizes the pathophysiology of rheumatoid arthritis (RA), its incidence and etiology, and conventional RA protein-based biomarkers. A brief overview of novel protein-based biomarkers and biomarkers with potential to be used in the diagnostics of seronegative RA is also provided. The core of the review resides in the comprehensive characterization of the glycosylation of immunoglobulins and their utility as novel biomarkers for the diagnostics of RA and seropositive RA. Two main approaches to the analysis of glycans present on immunoglobulins and other proteins are provided, including instrument-based and lectin-based approaches showing their clinical performance as RA biomarkers.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101633"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13571458/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148701745","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":"Simultaneous Spatial Mapping and Identification of C-Peptides Alongside Lipids in Mouse Pancreatic Tissues with Nano-DESI Mass Spectrometry Imaging.","authors":"Manxi Yang, Mushfeqa Iqfath, Frederick Nguele Meke, Zihan Qu, Emerson Hernly, Xindi Tang, Pei Su, Zhong-Yin Zhang, Julia Laskin","doi":"10.1016/j.mcpro.2026.101632","DOIUrl":"10.1016/j.mcpro.2026.101632","url":null,"abstract":"<p><p>Peptides secreted by pancreatic islet cells regulate glucose homeostasis. Their dysregulation may contribute to metabolic disorders such as diabetes and pancreatic disease. Here, we use nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) to identify and map intact and truncated C-peptides in mouse pancreatic tissue. Alongside commonly observed lipids and metabolites, we detected 36 multiply-charged acidic peptides that preferentially ionize in negative ion mode. On-tissue tandem mass spectrometry (MS/MS) was used to identify them as intact C-peptide isoforms and multiple truncated variants, including several previously unreported peptides. Molecular networking of MS/MS data assigned some unidentified species to C-peptide families based on shared fragmentation patterns. Ion images revealed distinct distributions of truncated peptides, with chemical gradients extending from β-cells into peri-insular acinar cells, suggesting their potential roles in endocrine-exocrine communication. These findings demonstrate the power of nano-DESI MSI to identify and spatially map novel peptides, which is critical for understanding their roles in pancreatic tissues.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101632"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13545366/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148648937","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":"Depletion-free Automated Enrichment of Serum Glycopeptides for High-Throughput Clinical Glycoproteomics.","authors":"Jung Hoon Choi, Geul Bang, Jong Hyun Seol, Seong Woo Choi, Su-Min Lee, Bao Tan Nguyen, Seungjin Na, Cheol-Jung Lee, Heeyoun Hwang, Jinsoo Chung, Sang-Jae Park, Sang Myoung Woo, Hye Gwang Jeong, Jin Young Kim","doi":"10.1016/j.mcpro.2026.101611","DOIUrl":"10.1016/j.mcpro.2026.101611","url":null,"abstract":"<p><p>High-throughput and reproducible enrichment of N-glycopeptides remains a major bottleneck in large-scale and clinical glycoproteomics, despite substantial advances in mass spectrometry. Here, we present a depletion-free, high-throughput N-glycopeptide enrichment workflow (3kMAX) that combines 3 kDa molecular weight cut-off filtration with positive-pressure-driven mixed anion exchange (MAX) enrichment in a 96-well format. This platform enables parallel processing of up to 96 samples with minimal operator intervention, improving analytical throughput, robustness and inter-batch reproducibility. Systematic evaluation using human serum demonstrated that the combined workflow achieved superior glycopeptide selectivity and identification depth compared with conventional manual MAX enrichment or molecular weight cut-off filtration alone, without introducing detectable glycan-type bias. Sensitivity analysis across a fourfold range of serum input volumes (5-20 μl) showed stable identification performance and quantitative reproducibility, with 10 μl identified as an optimal standardized input. Day-to-day reproducibility assessments further confirmed consistent quantitative performance across different acquisition densities. Application of the workflow to hepatocellular carcinoma serum samples enabled reproducible, large-scale quantitative glycoproteomic profiling and revealed extensive disease-associated glycosylation remodeling, including site-specific regulation within individual proteins. Collectively, the 3kMAX platform provides a scalable solution for site-specific serum N-glycoproteomics and facilitates its translation into clinical and biomarker-oriented applications.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101611"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13571447/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148331185","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":"A Scalable Low-Input Redox Proteomics Workflow Enables High-Throughput Analysis of Cysteine Oxidation in 96-Well Cell Cultures.","authors":"Adam M Kabza, Rosey Chu, Jesse Trejo, Xiaolu Li, Matthew J Gaffrey, David Hall, Wei-Jun Qian, Tong Zhang","doi":"10.1016/j.mcpro.2026.101641","DOIUrl":"10.1016/j.mcpro.2026.101641","url":null,"abstract":"<p><p>Reversible oxidation of cysteine residues (redox modifications) plays a crucial role in regulating protein function, signaling, and cellular homeostasis. These dynamic modifications act as molecular switches that transduce redox signals and modulate stress responses, metabolism, and pathogenesis. Redox proteomics enables systematic profiling of these modifications, quantifying the oxidation levels of tens of thousands of cysteine sites across the proteome and providing rich data to understand redox-regulated networks. However, conventional redox proteomic workflows are often limited by low-throughput and high sample requirements. Here, we present a high-throughput sample processing workflow for redox proteomics analysis from as little as 2 μg of protein. The workflow integrates 96-well plate-based cell culture, lysis, digestion, and cysteine-peptide enrichment, substantially increasing throughput and reducing hands-on processing time. Incorporating field asymmetric ion mobility spectrometry (FAIMS) further enhances redox proteome coverage by removing singly charged species in low-input samples, thereby increasing the signal of cysteine-containing peptides. Applying the workflow to RAW264.7 cells cultured in 96-well plates (40,000 cells per well), DIA identified >10,000 cysteine sites, and revealed a global increase in cysteine oxidation upon diamide treatment. To assess robustness, we repeated the 96-well experiment across three independent batches processed on different days and observed consistent coverage, reproducible quantification, and comparable diamide-induced oxidation of heat shock proteins, transcription factors, and protein kinases. Together, this workflow and new data acquisition scheme enable comprehensive redox proteomics from minimal inputs, paving the way for high-throughput sophisticated studies of redox modifications in cell signaling, disease, or large-scale screening of redox-modulating compounds.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101641"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13590100/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795533","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":"N-Glycoproteomics Sample Preparation Using Commercially Available Cellulose Magnetic Particles.","authors":"Ken Hanzawa, Miki Tanaka-Okamoto, Matej Nemcic, Yasuhide Miyamoto, Kazuki Nakajima","doi":"10.1016/j.mcpro.2026.101638","DOIUrl":"10.1016/j.mcpro.2026.101638","url":null,"abstract":"<p><p>Glycopeptide preparation is a critical step in bottom-up glycoproteomics. In this study, a robust method for N-glycopeptide preparation was developed using low-cost, commercially available magnetic particles. These particles function as a solid phase for hydrophilic interaction liquid chromatography (HILIC) to enrich N-glycopeptides from mixtures of N-glycopeptides and non-glycosylated peptides. Two types of magnetic particles, carboxylated polymer beads and cellulose resin, were compared. The latter demonstrated higher efficiency in N-glycopeptide recovery and non-glycopeptide removal. Biological samples often contain impurities such as salts and detergents that reduce efficiency and reproducibility of trypsin digestion and subsequent HILIC purification. To remove these impurities, we combined an optimized HILIC extraction protocol with a single-pot solid-phase-enhanced sample preparation protocol (SP3) for N-glycopeptide enrichment. Blood-derived samples (serum/plasma) were first tested. Compared with in-solution digestion, the method improved digestion efficiency and achieved N-glycopeptide recoveries comparable to those of conventional HILIC solid-phase extraction in a single tube. This method can be used for N-glycopeptide preparation not only from albumin-immunoglobulin-depleted serum but also from tissue extracts. Furthermore, the method has been successfully applied to glycoproteomic analysis of clinical serum and tissue samples from patients with gastric cancer, enabling detection of cancer-related alterations in glycoproteins.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101638"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13579034/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148713314","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":"An Integrated Cardiac Microtissue Proteome Map Extends Therapeutic Remodeling by Nanovesicles.","authors":"Jonathan Lozano, Jarmon G Lees, Jonathon Cross, Taha Aghajanzadeh, Haoyun Fang, Anne M Kong, Ren J Phang, Alice Pébay, Alin Rai, Shiang Y Lim, David W Greening","doi":"10.1016/j.mcpro.2026.101643","DOIUrl":"10.1016/j.mcpro.2026.101643","url":null,"abstract":"<p><p>Human cardiac microtissues are a promising model to study cardiac biology and disease, but their application is constrained by therapeutic remodeling strategies and limited knowledge of their functional protein expression profiles. Here, we define the use of human cardiac microtissue (hCMT) model generated by assembling induced pluripotent stem cell-derived endothelial cells, cardiac fibroblasts, and cardiomyocytes (CMs) to model ischemia-reperfusion injury (IRI) through a model of hypoxia and reoxygenation and nanovesicle (NV)-mediated functional remodeling. Engineered NVs, generated directly from human stem cells, have been shown to influence cardiac tissue and cell repair, and provide a platform for scalable and reproducible cell free-mediated therapy. We show the functional regulation of the hCMT model and define that administration of NVs (from human induced pluripotent stem cell origin) during reoxygenation significantly increase CM survival and preserve contractility function (contractile duration, relaxation time, and relaxation:contraction velocity). We establish NV uptake and transfer with target cells from the hCMT model. Quantitative proteomics was applied to decipher the cell proteome dynamics and molecular mechanisms of IRI in our in vitro model following NV treatment, linked with networks associated with cell survival, energy production, and stress response regulation. Notably, cell type-specific enrichment analysis revealed that NVs drive distinct proteomic remodeling based on their cell origin, where CERA NVs selectively upregulate cytoprotective and structural networks (such as HSP70, MYH6, and XIRP1) within parenchymal CMs, whereas CL2 NVs predominantly suppress non-myocyte activation and extracellular matrix remodeling factors within the endothelial and fibroblast compartments. Our findings provide an advanced human stem cell-based platform to understand underlying mechanisms of IRI and assess cell-free therapeutic cardioprotective strategies.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101643"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13602716/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795522","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":"Mass Spectrometry-Based De Novo Sequencing of N-Glycosylated Light Chains: Linking Urinary Proteoforms to In Situ Renal Amyloid Deposits.","authors":"Yueyue Zhu, Xin Wang, Xinyue Zhou, Shuang Wang, Peifeng Ji, Zidi Yan, Kai Chen, Xiaojuan Yu, Gang Liu, Li Yang, Yi Liu, Suxia Wang","doi":"10.1016/j.mcpro.2026.101652","DOIUrl":"10.1016/j.mcpro.2026.101652","url":null,"abstract":"<p><p>Systemic immunoglobulin light-chain (AL) amyloidosis is characterized by clonal immunoglobulin-secreting cells that produce a monoclonal light chain prone to misfolding and amyloid fibril formation in tissues. Understanding its molecular basis requires accurate full-length sequencing of amyloidogenic light chains and linkage of circulating light chains to renal deposits. Achieving this is technically challenging because the low abundance and N-glycosylation of amyloidogenic light chains can complicate protein purification and peptide-level sequence analysis. To address these challenges, we implemented a robust analytical pipeline that integrates intact-mass measurement by Q-TOF MS before and after deglycosylation with multi-protease digestion and de novo peptide sequencing to systematically characterize urinary N-glycosylated light chains. Mass shifts observed before and after deglycosylation supported the presence of N-glycosylation, whereas deglycosylated intact masses were used to constrain full-length sequence assembly. The assembled urinary light-chain sequences were subsequently compared with matched renal amyloid proteomes isolated by laser microdissection and analyzed by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enzymatic deglycosylation reduced glycan-induced spectral interference. Eight monoclonal light-chain sequences were assembled from urinary light chains, including five derived from the immunoglobulin kappa variable 1 (IGKV1), two from the immunoglobulin lambda variable 2 (IGLV2), and one from IGKV4. The close agreement between theoretical and experimental intact masses confirmed the accuracy and completeness of sequence assembly. In each renal amyloid proteome, the corresponding urine-derived sequence had the highest score among light-chain identifications of the patient's clinically determined isotype (κ or λ) and showed 87.6% to 100% variable-region peptide coverage. Together, these findings support urinary N-glycosylated monoclonal light chains as the precursor proteins of the corresponding renal amyloid fibrils. In conclusion, we have demonstrated a robust workflow that applies established de novo peptide sequencing to characterize urinary N-glycosylated light chains and trace the corresponding sequences in renal amyloid deposits, with potential applications across light chain-related diseases.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101652"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13635717/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148874564","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":"Dynamic Alterations of Site-Specific N-Glycoform and Plasma Proteasome Signatures Stratify Chemotherapy Benefit in Pancreatic Ductal Adenocarcinoma.","authors":"Shanying Gui, Yaqiong Zhang, Tao Xia, Jiazi Qian, Zemin Fang, Wenhui Ouyang, Jie Wang, Jie Chen, Xiaojun Ren, Jianxin Lyu, Wei Cui, Hezhi Fang","doi":"10.1016/j.mcpro.2026.101642","DOIUrl":"10.1016/j.mcpro.2026.101642","url":null,"abstract":"<p><p>Pancreatic ductal adenocarcinoma (PDAC) exhibits profound systemic remodeling during chemotherapy, yet how site-specific intact N-glycosylation in plasma is rewired by treatment remains largely undefined. Here, we perform an integrated plasma multi-omics study combining intact N-glycopeptidomics, deep proteomics, and metabolomics in a longitudinal cohort with paired pre- and post-chemotherapy samples and follow-ups (n = 177). Patients were further stratified according to radiographic treatment response. We identified 17,501 intact N-glycopeptides corresponding to 16,754 glycoforms, together with 6045 proteins and 1080 annotated metabolites. Paired analyses revealed extensive chemotherapy-associated changes across all three omics layers. Temporal glycopeptide and protein patterns were consistently enriched for complement, coagulation, extracellular-matrix, proteasome, and immune-related annotations, while oxidative-phosphorylation-related signatures showed relative depletion after treatment. Exploratory comparisons of within-patient changes (Δ = Post - Pre) between patients with partial response (PR) and those with stable or progressive disease highlighted 140 glycoforms, 18 glycosylation sites, and 103 proteins with response-associated patterns. These included coordinated increases in several proteasome-related plasma proteins in the PR group, as well as site- and glycoform-specific changes in complement- and acute-phase-related proteins. Response-associated differences in circulating metabolites further involved pyrimidine-, amino-acid-, and dipeptide-related features. Finally, leakage-controlled nested cross-validation prioritized 11 recurrent N-glycopeptide candidates and yielded modest prognostic discrimination, with a C-index of 0.591 and a 12-months area under the curve of 0.712. Collectively, this study provides a site- and glycoform-resolved view of chemotherapy-associated plasma molecular changes in PDAC and identifies candidate response- and prognosis-related features for future validation.</p>","PeriodicalId":18712,"journal":{"name":"Molecular & Cellular Proteomics","volume":" ","pages":"101642"},"PeriodicalIF":6.3,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13580066/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148795496","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}