Figure 1.

Schematic representation of the HCD spectral-aligning strategy for analysis of glycopeptides. Step 1, experimental b- and y-ions of deglycosylated peptide backbones were recorded from sample treated with PNGaseF. Step 2, these b- and y-ions of the deglycosylated peptides were used to screen the MS/MS spectra containing oxonium ions from the raw file for sample without PNGaseF treatment to identify putative glycopeptides. The putative glycopeptides were further evaluated by precursor ions, mass difference between glycopeptides and deglycosylated peptides, and Y-ions in the MS/MS spectra of the glycopeptides. Step 3, the identified MS/MS spectra of glycopeptides were used as spectral templates to identify other glycopeptides with the same peptide backbone but in different glycoforms.