{
  "abstract": "O-glycosylation is among the most abundant and structurally diverse post-translational modifications in eukaryotes, yet its heterogeneity renders O-glycoproteins exceptionally difficult to characterize. To overcome these challenges, we have developed an integrated mass spectrometry (MS) strategy to define the proteoform landscape of O-glycoproteins and applied it to human osteopontin (OPN). OPN is a disease-associated extracellular matrix protein subject to extensive modification. By combining native MS with serial exoglycosidase digestions, we directly resolved truncation, phosphorylation, sulfation, and O-glycosylation of OPN. Matched glycoproteomic analyses, using tailored (glyco)protease combinations, allowed us to quantify glycan heterogeneity inaccessible to conventional trypsin-based approaches or protein-centric methods. We integrated these datasets using forward compositional simulations to infer the intact OPN proteoform distribution and benchmarked the resulting models against an experimental intact-mass distribution obtained by proton-transfer charge-reduction MS. This comparison revealed that bottom-up O-glycoproteomics systematically underestimates the true extent of glycan sialylation, whereas assuming (near-)complete sialylation accurately reproduced the experimental intact OPN mass distribution. Together, these results provide a comprehensive, quantitative view of OPN compositional diversity and demonstrate how intact-protein and peptide-level measurements can be reconciled to resolve highly heterogeneous glycoform populations. The workflow establishes a broadly applicable framework for characterizing extensively O-glycosylated and multiply modified proteins.",
  "abstract_shown": true,
  "affiliations": [
    "University of Oxford"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "name": "K. C. Zouboulis"
    },
    {
      "name": "J. L. Bennett"
    },
    {
      "name": "L. A. Daly"
    },
    {
      "name": "S. A. Burnap"
    },
    {
      "name": "E. Holden"
    },
    {
      "name": "C. A. Lutomski"
    },
    {
      "name": "C. E. Eyers"
    },
    {
      "name": "C. V. Robinson"
    },
    {
      "name": "W. B. Struwe"
    },
    {
      "affs": [
        0
      ],
      "name": "J. L. P. Benesch"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/06pb2yws81j4z.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.07.749945",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1.full",
  "id": "06pb2yws81j4z",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biophysics"
    ],
    [
      "openalex",
      "1312|Glycosylation and Glycoproteins Research"
    ]
  ],
  "openalex": "W7212320454",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-07",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/06pb2yws81j4z.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:20:58Z",
      "native_id": "10.64898/2026.09.07.749945",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:biophysics",
      "subfield": "biophysics"
    }
  ],
  "title": "An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin",
  "title_text": "An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin",
  "type": "preprint",
  "updated_at": "2026-10-30T12:20:58Z",
  "url": "https://research.draconitic.com/paper/06pb2yws81j4z",
  "venue": "bioRxiv",
  "versions": []
}