Deciphering the O-glycosylation of HKU1 Spike Protein with the Dual-Functional Hydrophilic Interaction Chromatography Materials

HKU1 is a human betacoronavirus and infects host cells via highly glycosylated spike protein (S). At present, N-glycosylation of HKU1 S has been reported, however, little is known about its O-glycosylation, which hinders an in-depth understanding of its biological functions. Herein, a comprehensive study of O-glycosylation of HKU1 S was carried out based on dual-functional histidine-bonded silica materials (HBS). The enrichment method for O-glycopeptides with HBS was developed and validated using standard proteins. The application of the developed method to HKU1 S1 subunit resulted in 61 novel O-glycosylation sites, among which 56% were predicted to be exposed on protein outer surface. Moreover, the O-linked glycans and their abundance on each HKU1 S1 site were also analyzed. The obtained O-glycosylation dataset would provide valuable insights for the understanding of the structure of HKU1 S.