Ju Hwan Song (Korea Basic Science Institute)

Ju Yeon Lee (Korea Basic Science Institute)

Ju Hwan Song (Korea Basic Science Institute)

Sangeun Jang (Korea Basic Science Institute)

Jin-Woong Choi (Korea Basic Science Institute)

Seoyoung Hwang (Korea Basic Science Institute)

Kyoung Heon Kim (Korea University)

Hye-Yeon Kim (Korea Basic Science Institute)

Sun Cheol Park (Korea Basic Science Institute)

Wonbin Lee (Korea Basic Science Institute)

Ju Yeon Lee (Korea Basic Science Institute)

The COVID-19 pandemic, driven by SARS-CoV-2, continues to threaten global health as new variants emerge. The viral spike (S) protein and its human receptor ACE2 are heavily glycosylated, and these glycans modulate folding, receptor binding and immune recognition, making site-resolved glycoproteomic mapping essential. Here we profiled N- and O-glycosylation of recombinant spike RBD and ACE2 produced in Expi293F cells, and the S2 subunit expressed in <i style="font-family: " times="" new="" roman",="" serif;="" font-size:="" 11pt;"="">Nicotiana benthamiana</i>. Using an Orbitrap Eclipse Tribrid MS coupled to Ultimate 3000 RSLCnano and our Integrated GlycoProteome Analyzer (I-GPA), we identified 148 N- and 28 O-glycopeptides on RBD, 71 N-glycopeptides on S2, and 139 N-glycopeptides on ACE2. Novel PTMs—mannose-6-phosphate and GlcNAc-1-phosphate-6-O-mannose on RBD and ACE2 N-glycans, and O-acetylation on RBD O-glycans—were detected for the first time. Quantitative site-specific analysis revealed dominant occupancy at Asn331 (RBD), Asn1098 (S2) and Asn103 (ACE2); complex glycans prevailed on RBD and ACE2, whereas S2 was enriched in high-mannose structures. These data provide a high-resolution atlas of SARS-CoV-2 spike and ACE2 glycosylation, informing vaccine design and antibody- or lectin-based therapeutics.