Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

Yong Zhang; Wanjun Zhao; Yonghong Mao; Shisheng Wang; Yi Zhong; Tao Su; Meng Gong; Xiaofeng Lu; Jingqiu Cheng; Hao Yang

low-frequency glycosylation may occur, because our integrated methods, including glycopeptide enrichment and deglycosylation, failed to improve the spectra. Apart from the canonical N-glycosylation sequons, three non-canonical motifs of N-glycosites (N164, N334, and N536) involving N-X-C sequons were not glycosylated. Before enrichment, an average of 15 N-glycosites from trypsin-digested peptides and 13 N-glycosites from Glu-C-digested peptides were assigned. In contrast, hydrophilic enrichment resulted in a significant increase of these glycosites to 18 and 16, respectively (Table S1 ). To further assess the necessity for enrichment, we compared the representative spectra of one intact N-glycopeptide (N149) before and after enrichment. Without interference from the non-glycosylated peptides, the intact N-glycopeptide had more fragmented ions assigned to N-glycosites after enrichment ( Fig. S3 ). Complementary digestion with trypsin and Glu-C promoted the confident identification of four N-glycosites (N603, N616, N709 and N717) on two intact N-glycopeptides (Table S1 and Fig. S1C ). The introduction of Glu-C digestion resulted in the production of two short intact N-glycopeptides containing 23 and 36 amino acids, respectively. These peptides are more suitable for achieving better ionization and fragmentation than the long peptide of 48 and 57 amino acids obtained from trypsin digestion (Fig. S4 ). Deglycopeptides are suitable for verifying glycosylation sites (Fig. S5) . Unexpectedly, deglycopeptide peptides led to the loss of a few glycosites, presumably because of peptide loss during deglycosylation procedures. However, almost all glycosites were confidently confirmed using trypsin and Glu-C dual digestion (Table S1 ).