Co-eluting post-translational modification isoforms in collagen reveal fundamental challenge of LC-MS/MS site-specific analysis.

Collagen is a highly abundant extracellular matrix protein characterized by repetitive Gly-X-Y motifs and extensive post-translational modifications (PTMs), including hydroxylation of proline and lysine residues as well as O-glycosylation of hydroxylysine residues. This PTM density generates numerous peptide isoforms that challenge confident identification and quantification by LC-MS/MS. In this study, we have performed a systematic analysis of co-eluted, near-isobaric collagen peptides arising from site-specific PTM heterogeneity from the ECM of different mouse tissues. Using a publicly available healthy mouse tissue ECM proteomics dataset and a PTM-aware database search strategy, we demonstrate that near-isobaric collagen peptides frequently co-elute and co-fragment, producing chimeric spectra with high-confidence peptide-spectrum matches for multiple isoforms. Despite robust MS/MS evidence, overlapping retention times (RT) and identical m/z values prevent unambiguous localization of modification sites, necessitating exclusion from quantitative analyses. Our findings show that such co-elution reflects genuine biological heterogeneity rather than technical noise and highlight a critical gap between the complexity of collagen PTMs and current computational proteomics workflows, underscoring the need for advanced separation and PTM-aware analytical strategies.