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

To confirm site-specific N-glycan occupancy and exploit the potential impact on N-glycosylation by different protein sizes, recombinant RBDs (residues 319-541) from both human and insect cells were further analyzed (Table S6) The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.28.013276 doi: bioRxiv preprint 4B and 4C) are very close to those found in the S ectodomain and S1 subunit ( Fig. 3E and Fig. 3F ). The human cell-produced RBDs displayed more N-glycan compositions and complex glycan types, compared to insect cell-expressed proteins ( Fig. 4B and 4C ). Moreover, more than 80% of glycan compositions are identical at each site among different lengths of insect cell-expressed proteins (Fig. 4D) . Similarly, over 75% of the glycan compositions at each site were found to be shared by human cell-produced products (Fig. 4E ). The N-glycosylation of RBDs was verified by SDS-PAGE (Fig.S9) . These results suggest that the N-glycan compositions are conserved among different sizes of RBD proteins. Taken together, our data reveal the regular heterogeneity of N-glycan compositions at each site of the S protein subunits, primarily depending on host cells and glycosites. Intriguingly, the N-glycan types on S protein subunits are predominantly determined by host cells, regardless of the location of glycosites.