Fig. 1

Demonstration of the optimized MS/MS collision parameters for an intact glycopeptide. a Intact glycopeptide spectrum obtained using the optimized stepped-energy HCD-MS/MS method. b Spectrum obtained using the default single-energy HCD-MS/MS method for the same glycopeptide shown in a. The design of the upper box above each spectrum consists of the glycosylation site (glysite), modification (mod), spectrum name, precursor mass deviation, glycan composition and peptide sequence with ‘J’ indicating the N-glycosylation site. The glycan symbols are as follows: green circle for Hex, blue square for HexNAc and red triangle for fucose. Peak annotation is shown in the middle box: green and blue peaks represent the fragment ions of the glycan moiety or diagnostic glycan ions, and red peaks represent the Y ions from glycan fragmentation. For clarity, the scale of the relative intensity is automatically adjusted based on the highest peak between 700 and 2000 Th. Mass deviations of the annotated peaks are shown in the lower box