Characterization of intact sialylated glycopeptides and phosphorylated glycopeptides from IMAC enriched samples by EThcD fragmentation: Toward combining phosphoproteomics and glycoproteomics

Abstract

Protein phosphorylation and glycoprotein sialylation play a major role in regulating numerous cellular and molecular processes. Several previous studies demonstrated that it is possible to combine in-depth analysis of the phosphoproteome and sialome with common phosphopeptide enrichment methods that simultaneously enrich for sialylated glycopeptides. However, earlier workflows included enzymatic release of glycans and separation of phosphopeptides and formerly glycosylated peptides before independent LC–MS/MS analyses. Here, we demonstrate that electron-transfer/higher-energy collision dissociation (EThcD) enables identification of intact sialylated N-glycopeptides from IMAC enriched samples without additional sample preparation. Furthermore, this method allows for identification of intact glycopeptide sequences modified by phosphorylated glycans such as mannose-6-phosphate (M6P). This suggests that EThcD fragmentation of IMAC enriched samples provides a relatively straightforward means of implementing sialome and M6P-proteome analysis into existing phosphoproteomic workflows.