Proteomics analysis of site-specific glycoforms by a virtual multistage mass spectrometry method

Determination of site-specific glycoforms is the key to reveal the micro-heterogeneity of protein glycosylation at proteome level. Herein, we presented an integrated virtual multistage MS strategy to identify intact glycopeptides, which allowed the determination of site-specific glycoforms. In this strategy, the enzymatically de-glycosylated peptides and intact glycopeptides were mixed and analyzed in the same LC-MS/MS run. The acquired MS2 spectra of intact glycopeptides allowed determination of the glycans, and the MS2 spectra of the de-glycosylated peptides enabled the identification of peptide backbone sequences. Compared with the conventional multistage strategy, the peptide backbones could be directly identified by the MS2 of the de-glycopeptides with higher sensitivity. This strategy was first validated by analyzing the glycosites and site-specific glycoforms of mouse liver tissues. Then, it was applied to differential analysis of the glycoproteomes of hepatocellular carcinoma (HCC) and adjacent liver tissues. Compared with the identification scheme using only MS2 spectra of intact glycopeptides or glycosites, this approach enabled quantitative analysis on two levels, i.e. glycosites and site-specific glycoforms, simultaneously. Thus, it could be a powerful tool to characterize the subtle differences in the macro- and micro-heterogeneity of protein glycosylation for different samples.