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 expressed
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 on highly
enriched unlabeled O-glycopeptides with higher-energy collision dissociation
(HCD), electron-transfer/collision-induced dissociation (ETciD), and
electron transfer/higher-energy collisional dissociation (EThcD),
concluding that ETciD and EThcD with optimal supplemental activation
resulted in superior identification of glycopeptides and unambiguous
site localizations than HCD in a database search by Sequest HT. We
later described a pseudo-EThcD strategy that in silico concatenates the electron transfer dissociation spectrum with the
paired HCD spectrum acquired sequentially for the same precursor ions,
which combines the identification advantage of ETciD/EThcD with the
superior reporter ion quality of HCD. We demonstrated its improvements
in identification and quantification of isobaric mass tag-labeled
O-glycopeptides and showcased the discovery of the specific glycosites
of GalNAc transferase 11 (GALNT11) in HepG2 cells.