Ultraviolet Photodissociation Permits Comprehensive Characterization of O -Glycopeptides Cleaved with O -Glycoprotease IMPa.

Complete O -glycosite characterization, including identification of the peptides, localization of the glycosites, and mapping of the glycans, has been a persistent challenge in O -glycoproteomics owing to the technical challenges surrounding O -glycan analysis. Multi-glycosylated peptides pose an even greater challenge owing to their potential heterogeneity. Ultraviolet photodissociation (UVPD) can localize multiple post-translational modifications and is well-suited for the characterization of glycans. Three glycoproteins were assessed based on a strategy combining the use of O -glycoprotease IMPa and HCD-triggered UVPD for the complete characterization of O -glycopeptides. This approach localized multiple adjacent or proximal O -glycosites on individual glycopeptides and identified a previously unknown glycosite on etanercept at S218. Nine different glycoforms were characterized as a multi-glycosylated peptide from etanercept. The performance of UVPD was compared to that of HCD and EThcD for the localization of O -glycosites and the characterization of the constituent peptides and glycans.