An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin
- 1University of Oxford
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.
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BibTeX
@misc{zouboulis2026integrated,
title = {{An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin}},
author = {K. C. Zouboulis and J. L. Bennett and L. A. Daly and S. A. Burnap and E. Holden and C. A. Lutomski and C. E. Eyers and C. V. Robinson and W. B. Struwe and J. L. P. Benesch},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.07.749945},
url = {https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - An integrated mass spectrometry strategy for quantifying the proteoform diversity of the extensively modified O-glycoprotein Osteopontin
AU - K. C. Zouboulis
AU - J. L. Bennett
AU - L. A. Daly
AU - S. A. Burnap
AU - E. Holden
AU - C. A. Lutomski
AU - C. E. Eyers
AU - C. V. Robinson
AU - W. B. Struwe
AU - J. L. P. Benesch
PY - 2026
DA - 2026/09/07
PB - bioRxiv
DO - 10.64898/2026.09.07.749945
UR - https://www.biorxiv.org/content/10.64898/2026.09.07.749945v1
AB - 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.
ER -