Characterization of Intact Glycopeptides by a Combination of Hydrophilic Interaction Liquid Chromatography and Multiple Fragmentation Tandem Mass Spectrometry

Glycosylation of proteins plays a significant role in physiological and pathological processes. The general strategy to characterize the glycoprotein is to release the glycans from proteins, and then analyze them separately. However, the traditional methods suffer from inherent limitations since the relationship between proteins and glycans is neglected. Here we developed an integrated approach for intact glycopeptides analysis. Glycopeptides were first enriched selectively by home. made hydrophilic interaction liquid chromatography ( HILC) tips, and then analyzed by mass spectrometry. We applied multiple fragmentation methods including collision. induced dissociation ( CID), high energy collisional dissociation ( HCD) and electron transfer dissociation ( ETD) for comprehensive characterization of glycosylation. The results showed that the best ratio of sample vs material for glycopeptide enrichment was 1:50; CID with 35% collision energy cleaves glycosidic bonds, which was useful for glycan composition identification; HCD with 25% collision energy provided low mass diagnostic ions specific to glycan fragmentation, and also the specific Y1 fragment ion of glycopeptide; ETD cleaved peptide backbone, yielded peptide sequence and helps to assign glycosites. This work indicates that HILIC enrichment and multiple fragmentation techniques may be an effective way to characterize glycopeptides for the simultaneous elucidation of peptide sequence, glycosite and glycan structures.