A systematic evaluation of fragmentation methods for unlabeled and isobaric mass tag-labeled O-glycopeptides

Dissecting site-specific functions of O-glycosylation requires simultaneous identification and quantification of differentially ex-pressed O-glycopeptides by mass spectrometry. However, different dissociation methods have not been systematically compared in their performance in terms of identification, glycosite localization and quantification with isobaric labeling. Here, we conducted this comparison with higher-energy collision dissociation (HCD), electron-transfer/collision-induced dissociation (ETciD) and electron transfer/higher-energy collisional dissociation (EThcD), concluding that ETciD with optimal supplemental activation re-sulted in most identifications and unambiguous site localizations. We later described a pseudo EThcD strategy that in silico com-bines ETciD spectrum with HCD spectrum acquired sequentially for the same precursor, which combines the identification ad-vantage of ETciD with the superior reporter ion quality of HCD. We demonstrated its improvements in identifications and quantification of isobaric mass tag-labeled O-glycopeptides and showcased the discovery of the specific glycosylation sites of GalNAc tansferase 11 (GalNAc-T11) in HepG2 cells.