{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:58:37Z","timestamp":1790949517838,"version":"4.1.0"},"reference-count":46,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:00:00Z","timestamp":1789689600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:00:00Z","timestamp":1789689600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T00:00:00Z","timestamp":1789689600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"name":"PrognomiQ, Inc."}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Structural changes in protein glycosylation are recognized as phenotypes in numerous diseases, including cancer. Despite the promise of glycoproteomics for diagnostic biomarker discovery, large-scale studies remain limited by challenges in reproducibility, throughput, and quantitative precision. Here, we present a robust data-independent acquisition (DIA)-based plasma glycoproteomics platform that enables high-throughput and reproducible glycopeptide quantification suitable for large-cohort studies. The workflow integrates automated in-solution digestion and enrichment, optimized DIA-LC-MS acquisition, and a customized bioinformatics pipeline to achieve reproducible glycopeptide identification and quantitation. Across 560 replicates of a pooled human plasma sample processed in seven batches, the platform achieved high reproducibility, with intra-batch coefficients of variation (CVs) below 15% (n = 80 per batch) and an inter-batch CV of 22.9%. The method accurately captured expected fold changes from spiked-in glycoprotein standards. In the setting of a proof-of-concept pilot study to demonstrate the platform\u2019s ability to detect disease-associated glycosylation differences, the platform successfully identified glycoform-specific changes and distinguished between noncancer individuals (n = 39) and those with stage III\/IV lung cancer (n = 39). Together, these results establish a high-throughput DIA-based glycoproteomics workflow suitable for large-cohort studies to discover glycopeptide biomarker candidates.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00522","type":"journal-article","created":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T09:05:09Z","timestamp":1789722309000},"page":"5523-5533","source":"Crossref","is-referenced-by-count":0,"title":["A Robust DIA-Based\nPlatform for Large-Scale Plasma\nGlycoproteomics and Biomarker Discovery"],"prefix":"10.1021","volume":"25","author":[{"given":"Chi-Hung","family":"Lin","sequence":"first","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sayee","family":"Sawale","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark","family":"Marispini","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adam","family":"Poltorak","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joon-Yong","family":"Lee","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie","family":"Smith","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wan-Fang","family":"Chou","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Qian","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip","family":"Ma","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-8913-2588","authenticated-orcid":true,"given":"Bruce","family":"Wilcox","sequence":"additional","affiliation":[{"name":"PrognomiQ Inc. , , ,","place":["San Mateo, California, United States, 94403"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,18]]},"reference":[{"issue":"1","key":"2026100208413850700_cit1","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1093\/glycob\/cww086","article-title":"Biological\nRoles of Glycans","volume":"27","author":"Varki","year":"2017","journal-title":"Glycobiology"},{"issue":"4","key":"2026100208413850700_cit2","doi-asserted-by":"publisher","first-page":"421","DOI":"10.1016\/j.cbpa.2009.07.022","article-title":"Determination of Glycosylation Sites\nand Site-Specific Heterogeneity in Glycoproteins","volume":"13","author":"An","year":"2009","journal-title":"Curr. Opin. Chem. Biol"},{"issue":"10","key":"2026100208413850700_cit3","doi-asserted-by":"publisher","first-page":"1608","DOI":"10.1373\/clinchem.2008.108175","article-title":"High-Abundance\nPolypeptides of the\nHuman Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance","volume":"54","author":"Hortin","year":"2008","journal-title":"Clin. Chem"},{"issue":"21","key":"2026100208413850700_cit4","doi-asserted-by":"publisher","first-page":"17894","DOI":"10.1021\/acsomega.2c01193","article-title":"Aberrant Fucosylation\nof Saliva Glycoprotein Defining Lung Adenocarcinomas\nMalignancy","volume":"7","author":"Gao","year":"2022","journal-title":"ACS Omega"},{"key":"2026100208413850700_cit5","doi-asserted-by":"publisher","first-page":"100081","DOI":"10.1016\/j.mcpro.2021.100081","article-title":"In-Depth Site-Specific\nAnalysis of N-Glycoproteome in Human Cerebrospinal\nFluid and Glycosylation Landscape Changes in Alzheimer\u2019s Disease","volume":"20","author":"Chen","year":"2021","journal-title":"Mol. Cell. Proteomics"},{"key":"2026100208413850700_cit6","doi-asserted-by":"publisher","first-page":"53","DOI":"10.1016\/j.micres.2018.12.007","article-title":"Differential\nInvolvement of Glycans in the Binding of Staphylococcus Epidermidis\nand Corynebacterium Spp. to Human Sweat","volume":"220","author":"Lin","year":"2019","journal-title":"Microbiol.\nRes"},{"issue":"2","key":"2026100208413850700_cit7","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1373\/clinchem.2009.136333","article-title":"Sweetening the Pot:\nAdding Glycosylation to the Biomarker Discovery Equation","volume":"56","author":"Drake","year":"2010","journal-title":"Clin. Chem"},{"issue":"1","key":"2026100208413850700_cit8","doi-asserted-by":"publisher","first-page":"12","DOI":"10.1186\/s13045-024-01532-x","article-title":"Decoding the Glycoproteome:\nA New Frontier for Biomarker Discovery in Cancer","volume":"17","author":"He","year":"2024","journal-title":"J. Hematol. Oncol.J. Hematol Oncol"},{"issue":"7187","key":"2026100208413850700_cit9","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1038\/nature06916","article-title":"Mining the Plasma\nProteome for Cancer\nBiomarkers","volume":"452","author":"Hanash","year":"2008","journal-title":"Nature"},{"issue":"9","key":"2026100208413850700_cit10","doi-asserted-by":"publisher","first-page":"408.e9","DOI":"10.1016\/j.urolonc.2022.06.002","article-title":"Clinical Validation of IsoPSA, a\nSingle Parameter, Structure-Focused Assay for Improved Detection of\nProstate Cancer: A Prospective, Multicenter Study","volume":"40","author":"Klein","year":"2022","journal-title":"Urol. Oncol. Semin. Orig. Investig"},{"key":"2026100208413850700_cit11","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1016\/j.urology.2025.01.019","article-title":"Low Baseline\nIsoPSA Index Is Associated With a Prolonged Low Risk of Clinically\nSignificant Prostate Cancer Diagnosis in Men With an Elevated PSA","volume":"201","author":"Abdallah","year":"2025","journal-title":"Urology"},{"issue":"5","key":"2026100208413850700_cit12","doi-asserted-by":"publisher","first-page":"1983","DOI":"10.1002\/hep.30233","article-title":"Longitudinal\nAssessment of Three Serum Biomarkers to Detect Very Early-Stage Hepatocellular\nCarcinoma","volume":"69","author":"Choi","year":"2019","journal-title":"Hepatology"},{"issue":"2","key":"2026100208413850700_cit13","doi-asserted-by":"publisher","first-page":"470","DOI":"10.3390\/ijms18020470","article-title":"An Automated\nMicro-Total Immunoassay System for Measuring Cancer-Associated \u03912,3-Linked\nSialyl N-Glycan-Carrying Prostate-Specific Antigen May Improve the\nAccuracy of Prostate Cancer Diagnosis","volume":"18","author":"Ishikawa","year":"2017","journal-title":"Int.\nJ. Mol. Sci"},{"issue":"10","key":"2026100208413850700_cit14","doi-asserted-by":"publisher","first-page":"1716","DOI":"10.1038\/s41416-024-02644-4","article-title":"Diagnosing and Staging Epithelial\nOvarian Cancer by Serum Glycoproteomic Profiling","volume":"130","author":"Dhar","year":"2024","journal-title":"Br. J. Cancer"},{"issue":"11","key":"2026100208413850700_cit15","doi-asserted-by":"publisher","first-page":"1304","DOI":"10.1038\/s41592-021-01309-x","article-title":"Community\nEvaluation of Glycoproteomics Informatics Solutions Reveals High-Performance\nSearch Strategies for Serum Glycopeptide Analysis","volume":"18","author":"Kawahara","year":"2021","journal-title":"Nat. Methods"},{"issue":"6","key":"2026100208413850700_cit16","doi-asserted-by":"publisher","first-page":"3189","DOI":"10.1002\/jms.3189","article-title":"Quantitative Mapping of Glycoprotein\nMicro-heterogeneity and Macro-heterogeneity: An Evaluation of Mass\nSpectrometry Signal Strengths Using Synthetic Peptides and Glycopeptides","volume":"48","author":"Stavenhagen","year":"2013","journal-title":"J. Mass Spectrom"},{"key":"2026100208413850700_cit17","doi-asserted-by":"publisher","first-page":"100029","DOI":"10.1074\/mcp.R120.002277","article-title":"A Pragmatic Guide to Enrichment Strategies\nfor Mass Spectrometry\u2013Based Glycoproteomics","volume":"20","author":"Riley","year":"2021","journal-title":"Mol. Cell. Proteomics"},{"issue":"11","key":"2026100208413850700_cit18","doi-asserted-by":"publisher","first-page":"5478","DOI":"10.1021\/ac500945m","article-title":"Novel LC-MS2 Product Dependent\nParallel Data Acquisition Function and\nData Analysis Workflow for Sequencing and Identification of Intact\nGlycopeptides","volume":"86","author":"Wu","year":"2014","journal-title":"Anal. Chem"},{"issue":"8","key":"2026100208413850700_cit19","doi-asserted-by":"publisher","first-page":"3286","DOI":"10.1021\/acs.jproteome.0c00218","article-title":"Optimal Dissociation\nMethods Differ for N - and O -Glycopeptides","volume":"19","author":"Riley","year":"2020","journal-title":"J. Proteome Res"},{"issue":"1","key":"2026100208413850700_cit20","doi-asserted-by":"publisher","first-page":"bi1320s40","DOI":"10.1002\/0471250953.bi1320s40","article-title":"Byonic: Advanced\nPeptide and Protein\nIdentification Software","volume":"40","author":"Bern","year":"2012","journal-title":"Curr. Protoc. 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Rev"},{"key":"2026100208413850700_cit26","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1016\/j.eng.2025.01.011","article-title":"GlycoPro:\nA High-Throughput Sample-Processing\nPlatform for Multi-Glycosylation-Omics Analysis","volume":"57","author":"Liu","year":"2026","journal-title":"Engineering"},{"issue":"1","key":"2026100208413850700_cit27","doi-asserted-by":"publisher","first-page":"6483","DOI":"10.1038\/s41467-025-61673-6","article-title":"Library-Based Virtual Match-between-Runs\nQuantification in GlyPep-Quant Improves Site-Specific Glycan Identification","volume":"16","author":"Zhu","year":"2025","journal-title":"Nat. Commun"},{"issue":"11","key":"2026100208413850700_cit28","doi-asserted-by":"publisher","first-page":"2156","DOI":"10.1016\/j.ccell.2025.08.001","article-title":"Multi-Omic Profiling\nof Intraductal Papillary Neoplasms of the Pancreas Reveals Distinct\nPatterns and Potential Markers of Progression","volume":"43","author":"Wang","year":"2025","journal-title":"Cancer Cell"},{"issue":"20","key":"2026100208413850700_cit29","doi-asserted-by":"publisher","first-page":"7473","DOI":"10.1021\/acs.analchem.1c00645","article-title":"Automated Intact Glycopeptide\nEnrichment Method Facilitating Highly Reproducible Analysis of Serum\nSite-Specific N-Glycoproteome","volume":"93","author":"Liu","year":"2021","journal-title":"Anal. Chem"},{"issue":"8","key":"2026100208413850700_cit30","doi-asserted-by":"publisher","first-page":"100800","DOI":"10.1016\/j.mcpro.2024.100800","article-title":"Data-Independent\nAcquisition: A Milestone and Prospect in Clinical Mass Spectrometry\u2013Based\nProteomics","volume":"23","author":"Fro\u0308hlich","year":"2024","journal-title":"Mol. Cell. Proteomics"},{"issue":"6","key":"2026100208413850700_cit31","doi-asserted-by":"publisher","first-page":"922","DOI":"10.1002\/mas.21692","article-title":"Methods to Improve\nQuantitative Glycoprotein Coverage from Bottom-up\nLC-MS Data","volume":"41","author":"Chang","year":"2022","journal-title":"Mass Spectrom. Rev"},{"issue":"9","key":"2026100208413850700_cit32","doi-asserted-by":"publisher","first-page":"902","DOI":"10.1038\/s41592-019-0504-x","article-title":"Glyco-DIA: A Method for Quantitative\nO-Glycoproteomics with in Silico-Boosted Glycopeptide Libraries","volume":"16","author":"Ye","year":"2019","journal-title":"Nat. Methods"},{"key":"2026100208413850700_cit33","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1016\/j.jprot.2017.10.011","article-title":"Development\nof a\nData Independent Acquisition Mass Spectrometry Workflow to Enable\nGlycopeptide Analysis without Predefined Glycan Compositional Knowledge","volume":"172","author":"Lin","year":"2018","journal-title":"J. Proteomics"},{"issue":"5","key":"2026100208413850700_cit34","doi-asserted-by":"publisher","first-page":"959","DOI":"10.1002\/mas.70000","article-title":"Software Design and Analytical Challenges\nfor Confident Glycopeptide\nIdentification With Data-Independent Acquisition","volume":"45","author":"Nalehua","year":"2026","journal-title":"Mass Spectrom. Rev"},{"issue":"41","key":"2026100208413850700_cit35","doi-asserted-by":"publisher","first-page":"13774","DOI":"10.1021\/acs.analchem.1c01659","article-title":"Data-Independent Acquisition-Based\nMass Spectrometry (DIA-MS) for Quantitative Analysis of Intact N-Linked\nGlycopeptides","volume":"93","author":"Dong","year":"2021","journal-title":"Anal. Chem"},{"key":"2026100208413850700_cit36","doi-asserted-by":"publisher","first-page":"24300798","DOI":"10.1101\/2024.01.03.24300798","article-title":"Multi-Omics\nProfiling with Untargeted Proteomics for Blood-Based Early Detection\nof Lung Cancer","volume":"4","author":"Koh","year":"2024","journal-title":"Oncology"},{"issue":"7","key":"2026100208413850700_cit37","doi-asserted-by":"publisher","first-page":"966","DOI":"10.1093\/bioinformatics\/btq054","article-title":"Skyline: An Open Source Document Editor for Creating\nand Analyzing Targeted Proteomics Experiments","volume":"26","author":"MacLean","year":"2010","journal-title":"Bioinformatics"},{"issue":"4","key":"2026100208413850700_cit38","doi-asserted-by":"publisher","first-page":"1116","DOI":"10.1021\/acs.jproteome.5b00981","article-title":"Accounting\nfor the Multiple Natures of Missing Values in Label-Free Quantitative\nProteomics Data Sets to Compare Imputation Strategies","volume":"15","author":"Lazar","year":"2016","journal-title":"J. Proteome Res"},{"issue":"3","key":"2026100208413850700_cit39","doi-asserted-by":"publisher","first-page":"929","DOI":"10.1021\/acs.jproteome.3c00646","article-title":"timsTOF HT Improves Protein Identification and Quantitative\nReproducibility for Deep Unbiased Plasma Protein Biomarker Discovery","volume":"23","author":"Vitko","year":"2024","journal-title":"J. Proteome Res"},{"issue":"5","key":"2026100208413850700_cit40","doi-asserted-by":"publisher","first-page":"1941","DOI":"10.1021\/acs.jproteome.9b00753","article-title":"Quantitative\nLongitudinal Inventory of the N -Glycoproteome of\nHuman Milk from a Single Donor Reveals\nthe Highly Variable Repertoire and Dynamic Site-Specific Changes","volume":"19","author":"Zhu","year":"2020","journal-title":"J. Proteome Res"},{"issue":"10","key":"2026100208413850700_cit41","doi-asserted-by":"publisher","first-page":"3904","DOI":"10.1021\/acs.jproteome.6b00438","article-title":"Toward Automated N -Glycopeptide Identification\nin Glycoproteomics","volume":"15","author":"Lee","year":"2016","journal-title":"J. Proteome Res"},{"issue":"1","key":"2026100208413850700_cit42","doi-asserted-by":"publisher","first-page":"43","DOI":"10.1186\/s12014-022-09376-8","article-title":"Glycoproteomics\nRevealed Novel N-Glycosylation Biomarkers for Early Diagnosis of Lung\nAdenocarcinoma Cancers","volume":"19","author":"Fang","year":"2022","journal-title":"Clin. Proteomics"},{"issue":"1","key":"2026100208413850700_cit43","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1074\/mcp.M113.028621","article-title":"Integrated Glycoproteomics Demonstrates\nFucosylated Serum Paraoxonase 1 Alterations in Small Cell Lung Cancer","volume":"13","author":"Ahn","year":"2014","journal-title":"Mol. Cell. Proteomics"},{"issue":"7","key":"2026100208413850700_cit44","doi-asserted-by":"publisher","first-page":"2092","DOI":"10.1016\/j.celrep.2019.10.034","article-title":"Investigation\nof Proteomic and Phosphoproteomic Responses to Signaling Network Perturbations\nReveals Functional Pathway Organizations in Yeast","volume":"29","author":"Li","year":"2019","journal-title":"Cell Rep"},{"issue":"1","key":"2026100208413850700_cit45","doi-asserted-by":"publisher","first-page":"6073","DOI":"10.1038\/s41467-021-26246-3","article-title":"GproDIA Enables Data-Independent\nAcquisition Glycoproteomics with\nComprehensive Statistical Control","volume":"12","author":"Yang","year":"2021","journal-title":"Nat. Commun"},{"issue":"1","key":"2026100208413850700_cit46","doi-asserted-by":"publisher","first-page":"114","DOI":"10.1021\/acs.analchem.4c03069","article-title":"Quantitative\nSite-Specific\nGlycoproteomics Reveals Glyco-Signatures for Breast Cancer Diagnosis","volume":"97","author":"Yang","year":"2025","journal-title":"Anal. Chem"}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.6c00522","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5523\/68552552\/acs.jproteome.6c00522.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5523\/68552552\/acs.jproteome.6c00522.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T13:43:22Z","timestamp":1790948602000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/10\/5523\/5432516\/A-Robust-DIA-Based-Platform-for-Large-Scale-Plasma"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,18]]},"references-count":46,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,18]]},"published-print":{"date-parts":[[2026,10,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.6c00522","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,18]]}}}