Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV‑2 Strains

The coronavirus disease (COVID-19) pandemic, caused by
severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public
health globally. As the glycosylation of viral envelope glycoproteins
is strongly associated with their immunogenicity, intensive studies
have been conducted on the glycans of the glycoprotein of SARS-CoV-2,
the spike (S) protein. Here, we conducted intensive glycoproteomic
analyses of the SARS-CoV-2 S protein of ancestral and γ-variant
strains using a combinatorial approach with two different technologies:
mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based
glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic
search, identified 1448 (ancestral strain) and 1785 (γ-variant
strain) site-specific glycan compositions, respectively. Asparagine
at amino acid position 20 (N20) is mainly glycosylated within two
successive potential glycosylation sites, N17 and N20, of the γ-variant
S protein; however, we found low-frequency glycosylation at N17. Our
novel approaches, glycostem mapping and glycoleaf scoring, also illustrate
the moderately branched/extended, highly fucosylated, and less sialylated
natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized
the intensive end-capping of glycans by Lewis fucoses, which complemented
the glycoproteomic features. These results illustrate the high-resolution
glycoproteomic features of the SARS-CoV-2 S protein, contributing
to vaccine design and understanding of viral protein synthesis.