Fig. 1: O-glycosylation of SARS-CoV-2 S protein complies with the “O-Follow-N” rule.

a Schematic illustration of the S glycoprotein. The arrows underneath with blue numbers indicate the 22 identified N-glycosites. The branches above with numbers mark the 17 identified O-glycosites. T604/S605 was counted as one O-glycosite due to a lack of diagnostic ions. The determined O-glycosites are labeled in red color. NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; CH, central helix; CD, connector domain; TM, transmembrane domain; CT, cellular tail. b The composition of different O-glycoforms in S protein. HexNAcHexNeuAc(n) represents glycoforms with 1–3 NeuAc. c The identified O-glycopeptides of the S protein extracted from SARS-CoV-2 virions. The modified Asn and glycosylated S/T are marked in red color. b- or y- product ion fragments are marked as blue or red corners, respectively. c- or z- product ion fragments are annotated in orange or blue on the respective spectra. The diagnostic ions are labeled above and/or below the amino acid. T618 and S659 were identified in the sample with PNGase F de-N-glycosylation treatment, using Asn deamidation (+2.988261) and O-glycosylation as variable modifications for database search. S60, T124, S151, T236, T604/S605, T076, T1077, S1097 and T1100 were all identified in the sample without PNGase F treatment which were fragmented using SEC HCD. N- and O-glycosylation were searched simultaneously at the same peptide. d The overview of S and T sites of the S protein at N ± 1–3 positions flanking N-glycosites. 0 represents the position of glycosylated Asn. Identified O-glycosites are highlighted with orange color, and the determined sites are marked in red. e Structure-based display of the N- and O-glycosites in the S protein. Glycosites are marked on the top and side views of the structure of the trimeric SARS-CoV-2 S glycoprotein (PDB ID 6XR8). The N-glycosites are marked in green, while the O-glycosites at N ± 1, N ± 2, or N ± 3 are marked with red, blue, and brown, respectively. The surfaces of the S1 and S2 subunits are displayed in light and dark gray, respectively. f A bar graph showing the comparison of the relative O-glycosylation intensities on T618 in WT and N616Q S proteins. The relative intensity is calculated by normalizing the extracted chromatographic areas of O-glycopeptides containing T618 to the total extracted chromatographic areas of the sample. g Illustration of the O-glycosylation frequency at the proximal position associated to N-sequon.