Comprehensive N-glycosylation Profiling of Recombinant Spike S1 Protein from the Wild-type SARS-CoV-2 and Its Variants

Yao Peng¹Qiuyu Deng²Jiaqi Wu¹Lee-Fong Yau¹Yue Li²Jiaqi Qiu²Zhi-Hong Jiang¹Hudan Pan²Liu Liang^2*Jingrong Wang^2*Tian Tian TONG¹Youhua Xu^1*Manci Guan¹

• ¹Macau University of Science and Technology, Taipa, Macao, SAR China
• ²Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, China

By 2024, COVID-19 has become endemic, with new variants contributing to its continued spread. Herein, we performed an in-depth analysis of the N-glycosylation of the recombinant Spike S1 protein (S1 protein) across the Wild-type (WT) virus and its 5 variants, including Alpha, Beta, Gamma, Delta, and Lambda, by integrating ultrahigh performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) and unique TiO2-PGC Chip-based LC/MS techniques. A total of 332 glycan structures arising from 180 compositions on the S1 and RBD regions were identified, revealing remarkable glycosylation diversity of the S1 protein. Complex glycan was shown to be the dominant structure across variants. Neutral N-glycans are mainly di-antennary with two fucosyl groups, while the majority acidic N-glycans were multi-antennary with mono-fucosyl residues. In addition, sialic acid linkages of the N-glycans were extensively studied by utilizing 13 C-labeled standards and specific enzymes for the first time, showing the existence of both α-2,3 and α-2,6 linkages across WT and five variants. It should be noted that the Lambda variant shows more complex α-2,3 and α-2,6-linked glycans in the RBD region, which may potentially enhance its glycan shield effect. Acetylated glycans, which were identified on S protein for the first time, were found to be fully fucosylated on the S1 region and sialylated on the RBD region across all variants. UHPLC-TOF MS analysis revealed unoccupied N-glycosylation sites in S1-Gamma (N657), S1-Delta (N61), and S1-Lambda (N17, N61, N657), with N17 and N61 showing low glycan occupancy (0%-3.4%), suggesting these sites may lack of glycan shield protection. This study provides a comprehensive N-glycosylation profile of the S1 protein across different variants, offering an essential structural basis for future vaccine development and research on viral functions.• 27 acetylated N-glycans were identified on the Spike protein for the first time.• 51 acidic N-glycans with α-2,3 or α-2,6 linkages were identified, and their distribution across WT and five variants (Alpha, Beta, Gamma, Delta, and Lambda) in the S1 and RBD regions was comprehensively mapped for the first time. • Comprehensive glycosylation profiling was established for WT and five variants at the glycans and glycopeptide levels.