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However, conventional workflows that sequentially target distinct molecular classes are time- and resource-intensive, requiring repeated sequential sample preparation, imaging, and data integration. Here, we evaluate streamlined strategies for simultaneous or combined acquisition of N-glycan and collagen-derived peptide information using PNGase F and collagenase. In-solution studies demonstrate that simultaneous enzymatic digestion yields comparable peptide identifications and glycan profiles relative to traditional sequential workflows, with minimal impact on enzymatic specificity. On the basis of these findings, we developed and optimized MALDI-MSI protocols enabling either simultaneous enzyme application or sequential enzyme treatment with unified matrix deposition and single-pass imaging. While direct coapplication reduced image uniformity, a hybrid approach that used sequential enzyme deposition with combined imaging preserved spatial fidelity and spectral quality while significantly reducing processing and computational demands. Application to human tissues, including vertebral bone and ocular samples, highlights the utility of this workflow for fragile specimens and exploratory multiomic surveys. Collectively, these results establish a framework for integrated glycomic and proteomic imaging targeting the extracellular microenvironment, expanding multiomic MALDI-MSI analyses.<\/jats:p>","DOI":"10.1021\/jasms.6c00146","type":"journal-article","created":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T12:29:28Z","timestamp":1783513768000},"page":"1931-1941","source":"Crossref","is-referenced-by-count":0,"title":["Strategy for Simultaneous\nMultiomic Survey of N-Glycomic\nand Extracellular Matrix Proteome by Mass Spectrometry Imaging"],"prefix":"10.1021","volume":"37","author":[{"given":"Harrison B.","family":"Taylor","sequence":"first","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jade K.","family":"Macdonald","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3984-1574","authenticated-orcid":true,"given":"Walter K.","family":"Myers","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noah","family":"Bonnheim","sequence":"additional","affiliation":[{"name":"University of California San Francisco , , , ,","place":["San Francisco, California, United States, 94143"]},{"name":"Wake Forest University School of Medicine , , , ,","place":["Winston-Salem, North Carolina, United States, 27106"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9846-9389","authenticated-orcid":true,"given":"Anand S.","family":"Mehta","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard R.","family":"Drake","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0712-6533","authenticated-orcid":true,"given":"Christopher","family":"Hernandez","sequence":"additional","affiliation":[{"name":"University of California San Francisco , , , ,","place":["San Francisco, California, United States, 94143"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey C.","family":"Lotz","sequence":"additional","affiliation":[{"name":"University of California San Francisco , , , ,","place":["San Francisco, California, United States, 94143"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Fields","sequence":"additional","affiliation":[{"name":"University of California San Francisco , , , ,","place":["San Francisco, California, United States, 94143"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ba\u0308rbel","family":"Rohrer","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]},{"name":"Ralph H. John VA Medical Center , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4436-555X","authenticated-orcid":true,"given":"Peggi M.","family":"Angel","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]},{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,8]]},"reference":[{"key":"2026080510104397300_cit1","doi-asserted-by":"publisher","first-page":"2709","DOI":"10.1007\/s00216-020-03047-z","article-title":"Multiplexed imaging\nmass spectrometry of the extracellular matrix using serial enzyme\ndigests from formalin-fixed paraffin-embedded tissue sections","volume":"413","author":"Clift","year":"2021","journal-title":"Anal. Bioanal. Chem."},{"issue":"28","key":"2026080510104397300_cit2","doi-asserted-by":"publisher","first-page":"7011","DOI":"10.1007\/s00216-023-04983-2","article-title":"Evaluation of Antibody-Based\nSingle Cell Imaging Techniques Coupled to Multiplexed Imaging of N-Glycans\nand Collagen Peptides by Matrix-assisted Laser Desorption\/Ionization\nMass Spectrometry Imaging","volume":"415","author":"Dunne","year":"2023","journal-title":"Anal. Bioanal. Chem."},{"issue":"5","key":"2026080510104397300_cit3","doi-asserted-by":"publisher","first-page":"1148","DOI":"10.1021\/jasms.5c00032","article-title":"Maximizing\ndata coverage through eight sequential mass spectrometry images of\na single tissue section","volume":"36","author":"Seeley","year":"2025","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"1","key":"2026080510104397300_cit4","doi-asserted-by":"publisher","DOI":"10.1038\/s44303-024-00025-3","article-title":"Mass spectrometry imaging for spatially resolved multi-omics molecular\nmapping","volume":"2","author":"Zhang","year":"2024","journal-title":"Imaging"},{"issue":"12","key":"2026080510104397300_cit5","doi-asserted-by":"publisher","first-page":"6748","DOI":"10.3390\/ijms25126748","article-title":"Extracellular\nMicroenvironment Alterations in Ductal Carcinoma In Situ and Invasive\nBreast Cancer Pathologies by Multiplexed Spatial Proteomics","volume":"25","author":"Hulahan","year":"2024","journal-title":"Int. J. Mol. Sci."},{"issue":"20","key":"2026080510104397300_cit6","doi-asserted-by":"publisher","first-page":"9799","DOI":"10.1021\/ac402108x","article-title":"Matrix assisted\nlaser desorption ionization imaging mass spectrometry workflow for\nspatial profiling analysis of N-linked glycan expression in tissues","volume":"85","author":"Powers","year":"2013","journal-title":"Anal. Chem."},{"issue":"1","key":"2026080510104397300_cit7","doi-asserted-by":"publisher","first-page":"635","DOI":"10.1021\/acs.jproteome.7b00713","article-title":"Mapping Extracellular Matrix Proteins\nin Formalin-Fixed, Paraffin-embedded Tissues by MALDI Imaging Mass\nSpectrometry","volume":"17","author":"Angel","year":"2018","journal-title":"J. Proteome Res."},{"key":"2026080510104397300_cit8","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1007\/978-1-0716-1593-5_20","article-title":"Multiplexed Imaging\nMass Spectrometry of Histological Staining, N-glycan and Extracellular\nMatrix from One Tissue Section: A Tool for Fibrosis Research","volume":"2350","author":"Clift","year":"2021","journal-title":"Methods Mol. Biol."},{"key":"2026080510104397300_cit9","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwae043","article-title":"Spatial N-glycomics of the normal breast microenvironment\nreveal fucosylated and high-mannose N-glycan signatures related to\nBIRADS density and ancestry","volume":"34","author":"Rujchanarong","year":"2024","journal-title":"Glycobiology"},{"issue":"3","key":"2026080510104397300_cit10","doi-asserted-by":"publisher","first-page":"487","DOI":"10.1021\/jasms.3c00366","article-title":"Differential Protease\nSpecificity by Collagenase as a Novel Approach to Serum Proteomics\nThat Includes Identification of Extracellular Matrix Proteins Without\nEnrichment","volume":"35","author":"Macdonald","year":"2024","journal-title":"J. Am. Soc. Mass Spectrom."},{"issue":"12","key":"2026080510104397300_cit11","doi-asserted-by":"publisher","first-page":"2534","DOI":"10.1074\/mcp.TIR118.000900","article-title":"Online parallel accumulation\u2013serial fragmentation\n(PASEF) with a novel trapped ion mobility mass spectrometer","volume":"17","author":"Meier","year":"2018","journal-title":"Mol. Cell. Proteomics"},{"issue":"5","key":"2026080510104397300_cit12","doi-asserted-by":"publisher","first-page":"513","DOI":"10.1038\/nmeth.4256","article-title":"MSFragger: ultrafast and comprehensive\npeptide identification\nin mass spectrometry\u2013based proteomics","volume":"14","author":"Kong","year":"2017","journal-title":"Nat. Methods"},{"issue":"11","key":"2026080510104397300_cit13","doi-asserted-by":"publisher","first-page":"1885","DOI":"10.1007\/s13361-015-1204-0","article-title":"Novor: real-time\npeptide de novo sequencing software","volume":"26","author":"Ma","year":"2015","journal-title":"J. Am.\nSoc. Mass Spectrom."},{"issue":"17","key":"2026080510104397300_cit14","doi-asserted-by":"publisher","first-page":"4646","DOI":"10.1021\/ac0341261","article-title":"A statistical\nmodel for identifying proteins by tandem mass spectrometry","volume":"75","author":"Nesvizhskii","year":"2003","journal-title":"Anal. Chem."},{"issue":"20","key":"2026080510104397300_cit15","doi-asserted-by":"publisher","first-page":"5383","DOI":"10.1021\/ac025747h","article-title":"Empirical\nstatistical model to estimate the accuracy of peptide identifications\nmade by MS\/MS and database search","volume":"74","author":"Keller","year":"2002","journal-title":"Anal. Chem."},{"key":"2026080510104397300_cit16","first-page":"305","article-title":"Multiplexed and\nMultiomic Mass Spectrometry Imaging of MALDI-IHC Photocleavable Mass\nTag Tissue Probes, N-Glycomics, and the Extracellular Matrisome from\nFFPE Tissue Sections","volume-title":"Tissue Proteomics:\nMethods and Protocols","author":"Taylor","year":"2024"},{"issue":"1","key":"2026080510104397300_cit17","doi-asserted-by":"publisher","DOI":"10.1002\/cpps.68","article-title":"In Situ\nImaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or\nFormalin-Fixed Paraffin-Embedded Tissue","volume":"94","author":"Drake","year":"2018","journal-title":"Curr.\nProtoc. Protein Sci."},{"issue":"4","key":"2026080510104397300_cit18","doi-asserted-by":"publisher","first-page":"996","DOI":"10.1172\/JCI118884","article-title":"The human\nlumbar intervertebral disc: evidence for changes in the biosynthesis\nand denaturation of the extracellular matrix with growth, maturation,\nageing, and degeneration","volume":"98","author":"Antoniou","year":"1996","journal-title":"J. Clin. Invest."},{"issue":"1","key":"2026080510104397300_cit19","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1038\/s41413-025-00495-0","article-title":"Tissue and\nextracellular matrix remodeling of the subchondral bone during osteoarthritis\nof knee joints as revealed by spatial mass spectrometry imaging","volume":"14","author":"Schurman","year":"2026","journal-title":"Bone Res."},{"issue":"10","key":"2026080510104397300_cit20","doi-asserted-by":"publisher","first-page":"E592","DOI":"10.1097\/BRS.0000000000002432","article-title":"Cartilage\nendplate thickness variation measured by ultrashort echo-time MRI\nis associated with adjacent disc degeneration","volume":"43","author":"Berg-Johansen","year":"2018","journal-title":"Spine"},{"key":"2026080510104397300_cit21","doi-asserted-by":"publisher","first-page":"731","DOI":"10.3389\/fcell.2020.00731","article-title":"Distribution\nand function of glycosaminoglycans and proteoglycans in the development,\nhomeostasis and pathology of the ocular surface","volume":"8","author":"Puri","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"issue":"3","key":"2026080510104397300_cit22","doi-asserted-by":"publisher","first-page":"169","DOI":"10.3390\/biology10030169","article-title":"Extracellular matrix\nremodeling in the retina and optic nerve of a novel glaucoma mouse\nmodel","volume":"10","author":"Reinhard","year":"2021","journal-title":"Biology"},{"issue":"3","key":"2026080510104397300_cit23","doi-asserted-by":"publisher","first-page":"687","DOI":"10.3390\/cells10030687","article-title":"Cell\u2013matrix\ninteractions in the eye: from cornea to choroid","volume":"10","author":"Pouw","year":"2021","journal-title":"Cells"}],"container-title":["Journal of the American\nSociety for Mass Spectrometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jasms.6c00146","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/8\/1931\/65824171\/jasms.6c00146.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jamsef\/article-pdf\/37\/8\/1931\/65824171\/jasms.6c00146.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T14:22:08Z","timestamp":1785939728000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jamsef\/article\/37\/8\/1931\/5203341\/Strategy-for-Simultaneous-Multiomic-Survey-of-N"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,8]]},"references-count":23,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,7,8]]},"published-print":{"date-parts":[[2026,8,5]]}},"URL":"https:\/\/doi.org\/10.1021\/jasms.6c00146","relation":{},"ISSN":["1044-0305","1879-1123"],"issn-type":[{"value":"1044-0305","type":"print"},{"value":"1879-1123","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,8]]}}}