Simultaneous enrichment and sequential separation of O-linked glycopeptides and phosphopeptides with immobilized...

Protein phosphorylation and O-linked glycosylation are two critical posttranslational modifications (PTMs) and they both target identical amino acid residues, generating "crosstalk". Analysis of the crosstalk between these PTMs is crucial for deciphering their biological functions. Although several strategies have been established to enrich N-linked glycopeptides and phosphopeptides simultaneously, they are not appropriate for O-linked glycopeptides and phosphopeptides. In this study, we established a method for the simultaneous enrichment and sequential elution of O-linked glycopeptides and phosphopeptides into two fractions using commercially available immobilized titanium (IV) ion affinity chromatography (Ti⁴⁺-IMAC) materials. The established method exhibited high selectivity, repeatability, and recovery of the targeted peptides. Particularly, the overlap between the O-linked glycopeptide and phosphopeptide fractions was very low (∼3%). The application of this method to cell lysates of the colon cancer HT29 cell line resulted in a comparable number of enriched phosphopeptides as the coeluted method. However, the number of identified O-linked glycopeptides with our established method was 9.7-fold higher than that with the coelution method. Our study demonstrated that the established strategy was beneficial for the simultaneous analysis of O-linked glycopeptides and phosphopeptides, which might facilitate the study of the biological function of PTM crosstalk.