Hydrophobic-hydration hydrophilic interaction (H3I) capturing intact glycopeptides

H3I was adopted to idetify the glycosylation of bovine fetuin, human ACE2 and SARS-CoV-2 S1 proteins. Both N- and O-linked glycopeptides are successfully captured with significant enrichment rate improvements, especially for O-glycopeptides with relatively lower hydrophilicity. The majority of N- and O-glycosylation within SARS-CoV-2 S1 and hACE2 proteins are characterized simultaneously by H3I strategy, including 10 novel O-glycosylation regions with important functions in viral fusion and antibody evasion.