Comprehensive O-Glycosylation Analysis of the SARS-CoV-2 Spike Protein with Biomimetic Trp-Arg Materials

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious public health threat. Most vaccines against SARS-CoV-2 target the highly glycosylated spike protein (S). A good knowledge of the glycosylation profile of this protein is key to successful vaccine development. Unlike the 22 confirmed N-glycosylation sites on SARS-CoV-2 S, only a few O-glycosylation sites on this protein have been reported. This difference is mainly ascribed to the extremely low stoichiometry of O-glycosylation. Herein, we designed the biomimetic materials, Trp-Arg (WR) monomer-grafted silica microspheres (designated as WR-SiO2), and these biomimetic materials can enrich N- and O-linked glycopeptides with high selectivity. And WR-SiO2 can resist the nonglycopeptides' interference with the 100 molar fold of BSA during O-linked glycopeptide enrichment. We utilized WR-SiO2 to comprehensively analyze the O-glycosylation profile of recombinant SARS-CoV-2 S. Twenty-seven O-glycosylation sites including 18 unambiguous sites are identified on SARS-CoV-2 S. Our study demonstrates that the biomimetic polymer can offer specific selectivity for O-linked glycopeptides and pave the way for O-glycosylation research in biological fields. The O-glycosylation profile of SARS-CoV-2 S might supplement the comprehensive glycosylation in addition to N-glycosylation of SARS-CoV-2 S.

Automated summary

The COVID-19 pandemic, caused by SARS-CoV-2, poses a serious public health threat, and understanding the glycosylation profile of the spike protein is crucial for vaccine development. A biomimetic material, WR-SiO2, has been designed for selective enrichment of N- and O-linked glycopeptides, leading to the identification of 27 O-glycosylation sites on SARS-CoV-2 S. This study demonstrates the potential of biomimetic polymers for specific O-linked glycopeptide selectivity and advancement in O-glycosylation research.