{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T05:47:52Z","timestamp":1793339272815,"version":"4.5.0"},"reference-count":35,"publisher":"American Chemical Society (ACS)","issue":"31","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/na","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22225505"],"award-info":[{"award-number":["22225505"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22535002"],"award-info":[{"award-number":["22535002"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["W2412018"],"award-info":[{"award-number":["W2412018"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Cell surface glycoproteins are vital mediators of intercellular communication and primary targets for therapeutic intervention. However, the comprehensive profiling of the \u201csurface glycome\u201d on living cells remains hindered by the low abundance of these proteins and the inherent complexity of glycosylation. Here, we present GlycoCSP, a site-specific glycoproteomics strategy that utilizes alkyne-functionalized DNA scaffolds to achieve high-resolution GlycoBarcoding of cell surface proteins. By leveraging the spatial reach of extended DNA chains and a high-density alkyne array, GlycoCSP ensures specific labeling and robust covalent capture of surface glycoproteins. This platform integrates protein-level enrichment with an orthogonal tandem release proteolysis, enabling the precise mapping of N-glycosylation signatures at the site level. Applying GlycoCSP to live cells, we identified 2,016 extracellular N-glycosylation sites across 1,420 proteins, validated by deamidation mass shifts and the canonical N-X-S\/T\/C motif. Comparative analysis across breast cancer cell lines revealed that site-specific glycosylation occupancy provides a distinct layer of surfaceome heterogeneity that is independent of protein abundance. By providing a mass spectrometry-readable framework for decoding the surface glycode, GlycoCSP enables the unbiased discovery of glycosylation-dependent biomarkers and therapeutic targets previously inaccessible to conventional proteomics.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02112","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T08:23:46Z","timestamp":1785140626000},"page":"22887-22901","source":"Crossref","is-referenced-by-count":1,"title":["DNA-Alkyne-Chain\nProbes Enable N-Glycosite-Resolved\nGlycoBarcoding of Cell-Surface Proteins"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3115-9009","authenticated-orcid":true,"given":"Huimin","family":"Bao","sequence":"first","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yao","family":"Yao","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianpu","family":"Tang","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tailing","family":"Xue","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cao","family":"Fang","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2634-9281","authenticated-orcid":true,"given":"Dayong","family":"Yang","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P.R. China, 200438"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"issue":"7905","key":"2026081109103236500_cit1","doi-asserted-by":"publisher","first-page":"384","DOI":"10.1038\/s41586-022-04547-x","article-title":"Capturing a Rhodopsin\nReceptor Signalling Cascade Across a Native Membrane","volume":"604","author":"Chen","year":"2022","journal-title":"Nature"},{"issue":"10","key":"2026081109103236500_cit2","doi-asserted-by":"publisher","first-page":"713","DOI":"10.1038\/nchembio.437","article-title":"A Systematic Approach\nto Protein Glycosylation Analysis: A Path Through the Maze","volume":"6","author":"Marin\u0303o","year":"2010","journal-title":"Nat. Chem. Biol."},{"key":"2026081109103236500_cit3","doi-asserted-by":"crossref","unstructured":"Stark, J. C.; Gray, M. A.; Ibarlucea-Benitez, I.; Lustig, M.; Bond, A.; Cho, B.; Govil, I.; Luu, T.; Priestley, M. J.; Veth, T. S.; et al.\n          Antibody-Lectin Chimeras for Glyco-Immune\nCheckpoint Blockade.  Nat. Biotechnol.2025, 10.1038\/s41587-025-02884-6.","DOI":"10.1038\/s41587-025-02884-6"},{"issue":"4","key":"2026081109103236500_cit4","doi-asserted-by":"publisher","first-page":"378","DOI":"10.1038\/nbt.1532","article-title":"Mass-Spectrometric Identification and Relative Quantification\nof N-Linked Cell Surface Glycoproteins","volume":"27","author":"Wollscheid","year":"2009","journal-title":"Nat.\nBiotechnol."},{"issue":"8","key":"2026081109103236500_cit5","doi-asserted-by":"publisher","first-page":"4069","DOI":"10.1021\/acs.chemrev.4c00554","article-title":"Engineered\nProteins and Chemical Tools to Probe the Cell Surface Proteome","volume":"125","author":"Leung","year":"2025","journal-title":"Chem. Rev."},{"issue":"26","key":"2026081109103236500_cit6","doi-asserted-by":"publisher","first-page":"11008","DOI":"10.1073\/pnas.0704271104","article-title":"CD96 Is\na Leukemic\nStem Cell-Specific Marker in Human Acute Myeloid Leukemia","volume":"104","author":"Hosen","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"7","key":"2026081109103236500_cit7","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1038\/nrc1649","article-title":"The Sweet and Sour\nof Cancer: Glycans as Novel Therapeutic\nTargets","volume":"5","author":"Fuster","year":"2005","journal-title":"Nat. Rev. Cancer"},{"key":"2026081109103236500_cit8","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1186\/1741-7007-7-50","article-title":"Mapping the Human Membrane Proteome:\nA Majority of\nthe Human Membrane Proteins Can Be Classified According to Function\nand Evolutionary Origin","volume":"7","author":"Alme\u0301n","year":"2009","journal-title":"BMC Biol."},{"issue":"728","key":"2026081109103236500_cit9","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.adg3840","article-title":"The\nExtracellular Matrix as Hallmark of Cancer and Metastasis: From Biomechanics\nto Therapeutic Targets","volume":"16","author":"Sleeboom","year":"2024","journal-title":"Sci. Transl. Med."},{"key":"2026081109103236500_cit10","doi-asserted-by":"publisher","first-page":"958378","DOI":"10.3389\/fbinf.2022.958378","article-title":"The Druggable\nGenome: Twenty Years Later","volume":"2","author":"Radoux","year":"2022","journal-title":"Front. Bioinform."},{"issue":"46","key":"2026081109103236500_cit11","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1808790115","article-title":"The in Silico\nHuman Surfaceome","volume":"115","author":"Bausch-Fluck","year":"2018","journal-title":"Proc. Natl. Acad. Sci. U.\nS. A."},{"issue":"8","key":"2026081109103236500_cit12","doi-asserted-by":"publisher","first-page":"3235","DOI":"10.1021\/acs.jproteome.3c00800","article-title":"Veneer Is a Webtool for Rapid, Standardized,\nand Transparent Interpretation, Annotation, and Reporting of Mammalian\nCell Surface N-Glycocapture Data","volume":"23","author":"Luecke","year":"2024","journal-title":"J. Proteome\nRes."},{"issue":"6","key":"2026081109103236500_cit13","doi-asserted-by":"publisher","first-page":"660","DOI":"10.1038\/nbt827","article-title":"Identification\nand Quantification of N-Linked Glycoproteins Using Hydrazide Chemistry,\nStable Isotope Labeling and Mass Spectrometry","volume":"21","author":"Zhang","year":"2003","journal-title":"Nat. Biotechnol."},{"issue":"11","key":"2026081109103236500_cit14","doi-asserted-by":"publisher","first-page":"809","DOI":"10.1038\/nmeth.1392","article-title":"Enrichment of Glycopeptides for Glycan\nStructure and Attachment Site Identification","volume":"6","author":"Nilsson","year":"2009","journal-title":"Nat. Methods"},{"issue":"8","key":"2026081109103236500_cit15","doi-asserted-by":"publisher","first-page":"2085","DOI":"10.1074\/mcp.M114.045690","article-title":"A Combined\nOmics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation","volume":"14","author":"Graessel","year":"2015","journal-title":"Mol. Cell. Proteomics"},{"issue":"5","key":"2026081109103236500_cit16","doi-asserted-by":"publisher","first-page":"770","DOI":"10.1074\/mcp.M116.063859","article-title":"Monitoring\nCell-Surface N-Glycoproteome Dynamics by Quantitative Proteomics Reveals\nMechanistic Insights into Macrophage Differentiation","volume":"16","author":"Kalxdorf","year":"2017","journal-title":"Mol. Cell. Proteomics"},{"key":"2026081109103236500_cit17","doi-asserted-by":"publisher","first-page":"42610","DOI":"10.1038\/srep42610","article-title":"Identification of Extracellular Segments by Mass Spectrometry Improves\nTopology Prediction of Transmembrane Proteins","volume":"7","author":"Lango\u0301","year":"2017","journal-title":"Sci. Rep."},{"issue":"1","key":"2026081109103236500_cit18","doi-asserted-by":"publisher","first-page":"8880","DOI":"10.1038\/s41598-020-65831-2","article-title":"Partial\nProteolysis Improves the Identification of the Extracellular Segments\nof Transmembrane Proteins by Surface Biotinylation","volume":"10","author":"Lango\u0301","year":"2020","journal-title":"Sci. Rep."},{"issue":"47","key":"2026081109103236500_cit19","doi-asserted-by":"publisher","first-page":"17328","DOI":"10.1021\/acs.analchem.3c03626","article-title":"Sensitive and Specific Global Cell Surface N-Glycoproteomics\nShows Profound Differences Between Glycosylation Sites and Subcellular\nComponents","volume":"95","author":"de Haan","year":"2023","journal-title":"Anal. Chem."},{"issue":"17","key":"2026081109103236500_cit20","doi-asserted-by":"publisher","first-page":"4945","DOI":"10.1016\/j.bmcl.2011.05.045","article-title":"Cell Surface\nGlycoproteomic Analysis of Prostate Cancer-Derived PC-3 Cells","volume":"21","author":"Hubbard","year":"2011","journal-title":"Bioorg. Med. Chem. Lett."},{"issue":"36","key":"2026081109103236500_cit21","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202303410","article-title":"Click-iG: Simultaneous\nEnrichment and Profiling of Intact N-Linked,\nO-GalNAc, and O-GlcNAcylated Glycopeptides","volume":"62","author":"Liu","year":"2023","journal-title":"Angew. Chem., Int. Ed."},{"issue":"10","key":"2026081109103236500_cit22","doi-asserted-by":"publisher","first-page":"6934","DOI":"10.1021\/acs.analchem.9b01447","article-title":"Surface Glycoproteomic\nAnalysis Reveals That Both Unique and Differential Expression of Surface\nGlycoproteins Determine the Cell Type","volume":"91","author":"Suttapitugsakul","year":"2019","journal-title":"Anal.\nChem."},{"issue":"1","key":"2026081109103236500_cit23","doi-asserted-by":"publisher","first-page":"76","DOI":"10.1038\/s44161-022-00200-y","article-title":"Surfaceome Mapping of\nPrimary Human Heart Cells with CellSurfer Uncovers Cardiomyocyte Surface\nProtein LSMEM2 and Proteome Dynamics in Failing Hearts","volume":"2","author":"Luecke","year":"2023","journal-title":"Nat. Cardiovasc. Res."},{"issue":"2","key":"2026081109103236500_cit24","doi-asserted-by":"publisher","first-page":"695","DOI":"10.1073\/pnas.1813666116","article-title":"Multiomics of Azacitidine-Treated AML Cells Reveals\nVariable and Convergent Targets That Remodel the Cell-Surface Proteome","volume":"116","author":"Leung","year":"2019","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"23","key":"2026081109103236500_cit25","doi-asserted-by":"publisher","first-page":"7092","DOI":"10.1021\/jacs.7b13488","article-title":"The Metabolic Chemical\nReporter 6-Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity\nof O-GlcNAc Transferase and Catalyzes the Discovery of Intracellular\nProtein Modification by O-Glucose","volume":"140","author":"Darabedian","year":"2018","journal-title":"J. Am. Chem.\nSoc."},{"issue":"11","key":"2026081109103236500_cit26","doi-asserted-by":"publisher","first-page":"nwae341","DOI":"10.1093\/nsr\/nwae341","article-title":"Rapid and large-scale glycopeptide\nenrichment strategy based on chemical ligation","volume":"11","author":"Xiong","year":"2024","journal-title":"Natl. Sci. Rev."},{"issue":"1","key":"2026081109103236500_cit27","doi-asserted-by":"publisher","first-page":"44","DOI":"10.1038\/nprot.2008.211","article-title":"Systematic and integrative analysis\nof large gene lists using DAVID bioinformatics resources","volume":"4","author":"Huang","year":"2009","journal-title":"Nat. Protoc."},{"issue":"8","key":"2026081109103236500_cit28","doi-asserted-by":"publisher","first-page":"492","DOI":"10.1074\/mcp.O111.014704","article-title":"PaxDb, a Database\nof Protein Abundance Averages Across All Three Domains of Life*","volume":"11","author":"Wang","year":"2012","journal-title":"Mol. Cell. Proteomics"},{"issue":"12","key":"2026081109103236500_cit29","doi-asserted-by":"publisher","first-page":"1211","DOI":"10.1038\/nmeth.2646","article-title":"pLogo: A probabilistic\napproach to visualizing sequence\nmotifs","volume":"10","author":"O\u2019Shea","year":"2013","journal-title":"Nat. Methods"},{"issue":"6","key":"2026081109103236500_cit30","doi-asserted-by":"publisher","first-page":"884","DOI":"10.1093\/bioinformatics\/btt607","article-title":"Protter: Interactive\nprotein feature visualization and integration with experimental proteomic\ndata","volume":"30","author":"Omasits","year":"2014","journal-title":"Bioinformatics"},{"issue":"12","key":"2026081109103236500_cit31","doi-asserted-by":"publisher","first-page":"5460","DOI":"10.1038\/s41596-021-00621-2","article-title":"Rolling Circle Amplification\n(RCA)-Based DNA Hydrogel","volume":"16","author":"Yao","year":"2021","journal-title":"Nat. Protoc."},{"issue":"7","key":"2026081109103236500_cit32","doi-asserted-by":"publisher","first-page":"3422","DOI":"10.1021\/jacs.9b11001","article-title":"Double Rolling\nCircle Amplification Generates Physically\nCross-Linked DNA Network for Stem Cell Fishing","volume":"142","author":"Yao","year":"2020","journal-title":"J. Am. Chem. Soc."},{"issue":"8","key":"2026081109103236500_cit33","doi-asserted-by":"publisher","first-page":"475","DOI":"10.1038\/nchembio.1296","article-title":"Chemical Reporters\nfor Biological Discovery","volume":"9","author":"Grammel","year":"2013","journal-title":"Nat. Chem. Biol."},{"issue":"15","key":"2026081109103236500_cit34","doi-asserted-by":"publisher","first-page":"8439","DOI":"10.1073\/pnas.97.15.8439","article-title":"Internalization of\nCD26 by Mannose 6-Phosphate\/Insulin-Like Growth Factor II Receptor\nContributes to T Cell Activation","volume":"97","author":"Ikushima","year":"2000","journal-title":"Proc. Natl.\nAcad. Sci. U. S. A."},{"issue":"1","key":"2026081109103236500_cit35","doi-asserted-by":"publisher","first-page":"14570","DOI":"10.1038\/s41598-022-18779-4","article-title":"N-Glycosylation Is\nCrucial for Trafficking and Stability of SLC3A2 (CD98)","volume":"12","author":"Console","year":"2022","journal-title":"Sci. Rep."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c02112","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/31\/22887\/66276760\/acs.analchem.6c02112.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/31\/22887\/66276760\/acs.analchem.6c02112.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T13:24:37Z","timestamp":1786454677000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/31\/22887\/5233746\/DNA-Alkyne-Chain-Probes-Enable-N-Glycosite"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,27]]},"references-count":35,"journal-issue":{"issue":"31","published-online":{"date-parts":[[2026,7,27]]},"published-print":{"date-parts":[[2026,8,11]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c02112","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,27]]}}}