Glycosylation analysis of SARS-CoV-2 spike protein

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has globally impacted on public health. Since the glycosylation of viral envelope glycoproteins is deeply associated with their immunogenicity, intensive studies on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein, have been conducted. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and gamma variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1,448 (ancestral strain) and 1,785 (gamma variant strain) site-specific glycan compositions, respectively. An asparagine at amino acid position 20 (N20) was mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the gamma variant S protein; however, we newly found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrated the moderately branched/extended, highly fucosylated, and less sialylated nature of the glycoform of S proteins. The subsequent lectin microarray analysis emphasized intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and the understanding of viral protein synthesis.