Role of N343 glycosylation on the SARS-CoV-2 S RBD structure and co-receptor binding across variants of concern
- Department of Chemistry, Maynooth University, Ireland
- Department of Chemistry, University of Alberta, Canada
- Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Canada
- Département de Biochimie et Médecine Moléculaire, Université de Montréal, Canada
- School of Biological Sciences, University of Southampton, United Kingdom
Figures
Figure 1
Structure of the SARS-CoV-2 (WHu-1) Spike (S) glycoprotein and of the receptor binding domain (RBD) with a heat map of the interactions between the N343 glycan and the RBD in different variants of concern.
(a) Atomistic model of the SARS-CoV-2 (WHu-1) S glycoprotein trimer embedded in a lipid bilayer as reported in Casalino et al., 2020. In the conformation shown, the S bears the receptor binding …
Figure 2
Conformational changes of the S receptor binding domain (RBD) structure in function of loss of N-glycosylation at N343 in WHu-1, alpha and beta variants and binding affinities for interactions between different RBDs and GM1/2os.
(a) Kernel density estimates (KDE) plot of the backbone RMSD values calculated relative to frame 1 (t=0) of the trajectory for Region 1 (green) aa 337–353, Region 2 (yellow) aa 439–506, and Region 3 …
Figure 3
Conformational analysis and 3D structure of the S receptor binding domain (RBD) in the delta variant glycosylated at N343 with a diagram showing binding affinity to GM1/2os in the presence and absence of the glycan at N343 and in the omicron variant.
(a) Kernel density estimates (KDE) plot of the backbone RMSD values calculated relative to frame 1 (t=0) of the MD trajectories (MD1) trajectory for Region 1 (green) aa 337–353, Region 2 (yellow) aa …
Figure 4
Conformational dynamics of the BA.1 and BA.2.86 S receptor binding domain (RBD) in function of N343 glycosylation.
(a) Kernel density estimates (KDE) plot of the backbone RMSD values calculated relative to frame 1 (t=0) of the gaussian accelerated MD (GaMD) trajectory for Region 1 (green) aa 337–353, Region 2 …
Appendix 1—figure 1
Amino acid sequence of SARS-CoV-2 spike (S) protein variants and mutations on receptor binding domain (RBD) used in this study.
Appendix 1—figure 2
Conformational dynamics of the WHu-1 receptor binding domain (RBD) in function of N343 glycosylation.
(a) Kernel density estimates (KDE) plots of the backbone RMSD values calculated relative to frame 1 (t=0) of the gaussian accelerated MD (GaMD) trajectory for Region 1 (green) aa 337–353, Region 2 …
Appendix 1—figure 3
Bar plot of the interaction frequencies (%) of the N343 N-glycan with the different residues within the aa 365–375 loop for each variants of concern (VoC).
The interactions include both hydrogen bonding and dispersion (van der Waals) contacts.
Appendix 1—figure 4
Conformational dynamics of the delta receptor binding domain (RBD) in function of N343 glycosylation.
(a) Kernel density estimates (KDE) plot of the backbone RMSD values calculated relative to frame 1 (t=0) of the MD1 trajectory for Region 1 (green) aa 337–353, Region 2 (yellow) aa 439–506, and …
Appendix 1—figure 5
Relative abundance of N-glycans at N331 and N343 on the wild-type (WT) receptor binding domain (RBD) before and after endoF3 treatment.
Appendix 1—figure 6
Conformational dynamics of the BA.1 receptor binding domain (RBD) in function of N343 glycosylation.
(a) Kernel density estimates (KDE) plot of the backbone RMSD values calculated relative to frame 1 (t=0) of the MD1 (left) and MD2 (right) trajectories for Region 1 (green) aa 337–353, Region 2 …
Tables
Appendix 1—table 1
Molecular dynamics (MD) sampling methods and corresponding times in the production phase of the simulations.
MD1 and MD2 indicate trajectories collected through conventional (deterministic) sampling. Gaussian accelerated MD (GaMD) indicates trajectories obtained through Gaussian accelerated MD sampling. …
| N343-FA2G2 (μs) | N343 NoGly (μs) | |||||
|---|---|---|---|---|---|---|
| Variant | MD1 | MD2 | GaMD | MD1 | MD2 | GaMD |
| WHu-1 | 2.5 | 2.0 | 2.0 | 3.0 | 2.0 | 2.0 |
| Alpha (B.1.1.7) | 2.0 | - | - | - | 2.0 | - |
| Beta (B.1.351) | 2.0 | - | - | - | 2.0 | - |
| Delta (B.1.617.2) | 2.0 | 2.0 | 2.0 | 3.0 | - | 2.0 |
| Omicron (BA.1) | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 |
| Omicron (BA.2.86) | 2.0 | - | - | - | - | - |
| Total (46.5) | 12.5 | 6.0 | 6.0 | 8.0 | 8.0 | 6.0 |
Appendix 1—table 2
Affinities of the oligosaccharides of GM1 and GM2 (GM1os and GM2os, respectively) for WHu-1, Alpha, Beta, Delta, and Omicron receptor binding domain (RBD) and endoH-treated WHu-1 RBD and endoF3-treated WHu-1 and Delta RBD measured by ESI-MS for aqueous ammonium acetate (100 mM, pH 7.4) solutions containing a given RBD (5 μM) and glycan (three different initial concentrations ranging from 10 μM to 150 μM).
Data represent mean ± SD; n = 3 independent experiments for each glycan concentration.
| Variant | GM1os Kd (mM) | GM2os Kd (mM) |
|---|---|---|
| WHu-1 RBD (sample 1; HEK293-a) | 0.16 ± 0.04 | 0.17 ± 0.02 |
| WHu-1 RBD (sample 2; HEK293-a) | 0.18 ± 0.01 | 0.22 ± 0.03 |
| WHu-1 RBD (sample 3; HEK293-b) | 0.07 ± 0.01 | 0.09 ± 0.02 |
| endoF3-treated WHu-1 RBD (HEK293-b) | 3.6 ± 0.7 | 5.7 ± 0.6 |
| alpha (HEK293-b) | 0.15 ± 0.03 | 0.16 ± 0.03 |
| beta (HEK293-b) | 1.3 ± 0.4 | 1.10 ± 0.2 |
| delta (HEK293-b) | 0.11 ± 0.01 | 0.08 ± 0.02 |
| endoF3-treated delta (HEK293-b) | 0.12 ± 0.01 | 0.06 ± 0.01 |
| micron (HEK293-b) | 1.1 ± 0.2 | 0.70 ± 0.16 |
Appendix 1—table 3
Summary of molecular weights (MWs) of WHu-1 receptor binding domain (RBD) glycoforms (WTx) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) |
|---|---|---|---|---|---|
| WT1 | 31342.4 | 25.5 | 9_11_2_0 | 31345.1 | 5.3 |
| WT2 | 31402.4 | 2.6 | 11_9_1_1 | 31408.0 | 2.4 |
| WT3 | 31505.2 | 42.7 | 9_9_0_3 | 31520.1 | 6.9 |
| WT4 | 31527.0 | 34.3 | 10_9_1_2 | 31537.1 | 2.1 |
| WT5 | 31609.4 | 19.0 | 11_10_3_0 | 31612.1 | 5.3 |
| WT6 | 31668.8 | 48.8 | 10_9_0_3 | 31682.1 | 5.4 |
| WT7 | 31690.2 | 56.2 | 11_9_1_2 | 31699.1 | 1.0 |
| WT8 | 31711.4 | 17.8 | 9_10_0_3 | 31723.2 | 3.7 |
| WT9 | 31771.1 | 43.9 | 12_10_3_0 | 31774.2 | 4.9 |
| WT10 | 31794.0 | 65.6 | 10_11_0_2 | 31797.2 | 4.8 |
| WT11 | 31816.5 | 26.5 | 10_9_1_3 | 31828.2 | 3.7 |
| WT12 | 31835.3 | 30.6 | 11_9_2_2 | 31845.2 | 1.9 |
| WT13 | 31854.4 | 44.6 | 9_10_1_3 | 31869.2 | 6.8 |
| WT14 | 31875.2 | 47.5 | 10_10_2_2 | 31886.2 | 3.1 |
| WT15 | 31896.4 | 35.8 | 11_10_1_2 | 31902.2 | 2.2 |
| WT16 | 31935.8 | 5.6 | 10_11_1_2 | 31943.3 | 0.6 |
| WT17 | 31957.8 | 85.0 | 11_11_0_2 | 31959.2 | 6.6 |
| WT18 | 31979.7 | 98.1 | 13_11_0_1 | 31992.3 | 4.6 |
| WT19 | 31001.2 | 61.3 | 9_10_2_3 | 32015.3 | 6.1 |
| WT20 | 32038.7 | 17.3 | 11_10_0_3 | 32047.3 | 0.6 |
| WT21 | 32060.1 | 55.7 | 12_10_1_2 | 32064.3 | 3.9 |
| WT22 | 32083.3 | 55.9 | 10_11_0_3 | 32088.3 | 3.0 |
| WT23 | 32105.5 | 1.1 | 12_11_0_2 | 32121.3 | 7.8 |
| WT24 | 32125.1 | 12.6 | 13_11_1_1 | 32138.3 | 5.2 |
| WT25 | 32144.3 | 72.6 | 10_12_1_2 | 32146.3 | 5.9 |
| WT26 | 32165.0 | 63.5 | 12_12_1_1 | 32179.3 | 6.4 |
| WT27 | 32185.7 | 39.8 | 11_10_1_3 | 32193.3 | 0.4 |
| WT28 | 32247.8 | 88.4 | 11_11_0_3 | 32250.3 | 5.5 |
| WT29 | 32270.3 | 100.0 | 13_11_0_2 | 32283.4 | 5.1 |
| WT30 | 32291.8 | 62.4 | 12_14_1_0 | 32294.4 | 5.4 |
| WT31 | 32348.8 | 58.7 | 12_10_1_3 | 32355.4 | 1.4 |
| WT32 | 32371.5 | 46.6 | 10_11_0_4 | 32379.4 | 0.1 |
| WT33 | 32434.4 | 69.7 | 10_12_1_3 | 32437.4 | 5.0 |
| WT34 | 32456.1 | 76.4 | 12_12_1_2 | 32470.4 | 6.3 |
| WT35 | 32477.0 | 43.6 | 11_10_1_4 | 32484.4 | 0.6 |
| WT36 | 32538.2 | 78.0 | 11_11_0_4 | 32541.4 | 4.8 |
| WT37 | 32560.9 | 79.6 | 13_11_0_3 | 32574.4 | 5.6 |
| WT38 | 32583.0 | 49.5 | 12_14_1_1 | 32585.5 | 5.5 |
| WT39 | 32615.2 | 19.1 | 11_10_0_5 | 32629.5 | 6.3 |
| WT40 | 32637.9 | 60.5 | 12_10_1_4 | 32646.5 | 0.6 |
| WT41 | 32659.6 | 39.0 | 10_11_0_5 | 32670.5 | 2.9 |
| WT42 | 32725.4 | 55.2 | 10_12_1_4 | 32728.5 | 4.9 |
| WT43 | 32747.7 | 58.2 | 12_12_1_3 | 32761.5 | 5.8 |
| WT44 | 32768.8 | 4.1 | 11_10_1_5 | 32775.5 | 1.3 |
| WT45 | 32826.8 | 1.7 | 11_11_0_5 | 32832.5 | 2.3 |
| WT46 | 32851.5 | 10.7 | 13_11_0_4 | 32865.5 | 6.1 |
| WT47 | 32873.7 | 4.3 | 12_14_1_2 | 32876.6 | 5.1 |
| WT48 | 32927.5 | 49.5 | 12_10_1_5 | 32937.6 | 2.1 |
| WT49 | 32949.8 | 15.6 | 10_11_0_6 | 32961.6 | 3.8 |
| WT50 | 33293.5 | 34.6 | 13_11_1_5 | 33302.7 | 1.2 |
| WT51 | 33583.8 | 9.4 | 13_11_1_6 | 33593.8 | 2.0 |
Appendix 1—table 4
Summary of molecular weights (MWs) of B.1.1.7 receptor binding domain (RBD) glycoforms (Alpha, Ax) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) |
|---|---|---|---|---|---|
| A1 | 30894.6 | 6.4 | 9_10_0_0 | 30899.0 | 4.3 |
| A2 | 31080.7 | 17.1 | 9_8_2_1 | 31076.0 | 4.7 |
| A3 | 31137.8 | 5.3 | 9_9_3_0 | 31134.1 | 3.7 |
| A4 | 31160.2 | 3.2 | 9_7_2_2 | 31164.0 | 3.8 |
| A5 | 31184.9 | 3.7 | 9_10_0_1 | 31190.1 | 5.2 |
| A6 | 31243.1 | 13.0 | 10_8_2_1 | 31238.1 | 5.1 |
| A7 | 31265.9 | 10.9 | 10_11_0_0 | 31264.1 | 1.8 |
| A8 | 31300.5 | 4.5 | 10_9_3_0 | 31296.1 | 4.4 |
| A9 | 31319.6 | 3.4 | 8_10_2_1 | 31320.1 | 0.5 |
| A10 | 31346.6 | 2.6 | 10_10_0_1 | 31352.1 | 5.5 |
| A11 | 31369.5 | 19.4 | 11_10_1_0 | 31369.1 | 0.4 |
| A12 | 31405.1 | 26.4 | 8_9_2_2 | 31408.1 | 3.0 |
| A13 | 31427.3 | 13.6 | 11_11_0_0 | 31426.1 | 1.1 |
| A14 | 31449.4 | 4.9 | 7_10_4_1 | 31450.2 | 0.8 |
| A15 | 31482.0 | 4.6 | 9_10_2_1 | 31482.2 | 0.1 |
| A16 | 31505.7 | 3.4 | 7_11_3_1 | 31507.2 | 1.5 |
| A17 | 31529.1 | 30.9 | 12_10_1_0 | 31531.2 | 2.0 |
| A18 | 31553.5 | 35.3 | 10_11_0_1 | 31555.2 | 1.7 |
| A19 | 31590.8 | 27.8 | 12_11_0_0 | 31588.2 | 2.6 |
| A20 | 31611.6 | 16.5 | 8_10_2_2 | 31611.2 | 0.4 |
| A21 | 31658.9 | 9.8 | 11_10_3_0 | 31661.2 | 2.3 |
| A22 | 31693.9 | 8.8 | 13_10_1_0 | 31693.2 | 0.6 |
| A23 | 31715.2 | 96.9 | 11_11_0_1 | 31717.2 | 2.0 |
| A24 | 31737.6 | 13.7 | 12_11_1_0 | 31734.3 | 3.3 |
| A25 | 31773.3 | 19.8 | 9_10_2_2 | 31773.3 | 0.1 |
| A26 | 31793.9 | 13.4 | 12_12_0_0 | 31791.3 | 2.7 |
| A27 | 31819.9 | 31.4 | 12_10_1_1 | 31822.3 | 2.4 |
| A28 | 31844.4 | 20.4 | 10_11_0_2 | 31846.3 | 1.9 |
| A29 | 31880.1 | 13.3 | 12_11_0_1 | 31879.3 | 0.8 |
| A30 | 31901.1 | 78.6 | 8_10_4_2 | 31903.3 | 2.3 |
| A31 | 31920.8 | 4.8 | 11_12_0_1 | 31920.3 | 0.5 |
| A32 | 31937.3 | 2.7 | 12_12_1_0 | 31937.3 | 0.1 |
| A33 | 31957.6 | 10.8 | 13_12_0_0 | 31953.3 | 4.2 |
| A34 | 31978.1 | 10.0 | 9_11_2_2 | 31976.3 | 1.7 |
| A35 | 31006.1 | 100.0 | 11_11_0_2 | 31008.3 | 2.2 |
| A36 | 32027.7 | 5.2 | 12_11_3_0 | 32026.4 | 1.3 |
| A37 | 32061.8 | 4.9 | 9_10_2_3 | 32064.4 | 2.6 |
| A38 | 32081.4 | 53.5 | 12_12_0_1 | 32082.4 | 1.0 |
| A39 | 32109.6 | 10.5 | 12_10_1_2 | 32113.4 | 3.8 |
| A40 | 32137.5 | 13.8 | 10_11_2_2 | 32138.4 | 0.9 |
| A41 | 32191.8 | 82.8 | 13_11_3_0 | 32188.4 | 3.4 |
| A42 | 32212.7 | 3.2 | 11_12_0_2 | 32211.4 | 1.3 |
| A43 | 32227.5 | 2.6 | 12_12_1_1 | 32228.4 | 1.0 |
| A44 | 32266.5 | 39.1 | 9_11_2_3 | 32267.4 | 0.9 |
| A45 | 32297.9 | 48.2 | 11_11_0_3 | 32299.4 | 1.5 |
| A46 | 32372.0 | 53.5 | 12_12_0_2 | 32373.5 | 1.5 |
| A47 | 32393.9 | 5.0 | 11_10_4_2 | 32389.5 | 4.4 |
| A48 | 32446.4 | 31.2 | 11_11_1_3 | 32445.5 | 0.9 |
| A49 | 32483.1 | 35.2 | 13_11_3_1 | 32479.5 | 3.6 |
| A50 | 32504.3 | 4.7 | 11_12_2_2 | 32503.5 | 0.7 |
| A51 | 32557.2 | 43.9 | 14_12_3_0 | 32553.6 | 3.7 |
| A52 | 32590.0 | 10.0 | 11_11_2_3 | 32591.5 | 1.6 |
| A53 | 32631.8 | 25.2 | 10_12_2_3 | 32632.6 | 0.8 |
| A54 | 32663.5 | 34.6 | 12_12_0_3 | 32664.6 | 1.1 |
| A55 | 32707.1 | 4.5 | 11_13_2_2 | 32706.6 | 0.5 |
| A56 | 32737.2 | 44.7 | 11_11_1_4 | 32736.6 | 0.6 |
| A57 | 32774.4 | 6.2 | 13_11_3_2 | 32770.6 | 3.8 |
| A58 | 32811.6 | 21.6 | 12_12_1_3 | 32810.6 | 0.9 |
| A59 | 32848.2 | 24.6 | 14_12_3_1 | 32844.7 | 3.6 |
| A60 | 32869.8 | 4.8 | 12_13_2_2 | 32868.7 | 1.2 |
| A61 | 32887.8 | 3.5 | 13_13_1_2 | 32884.7 | 3.1 |
| A62 | 32922.7 | 34.7 | 10_12_2_4 | 32923.7 | 1.0 |
| A63 | 32954.7 | 11.5 | 12_12_0_4 | 32955.7 | 1.0 |
| A64 | 32998.0 | 16.2 | 11_13_2_3 | 32997.7 | 0.3 |
| A65 | 33028.4 | 28.5 | 11_11_1_5 | 33027.7 | 0.7 |
| A66 | 33050.4 | 3.3 | 12_11_4_3 | 33045.7 | 4.7 |
| A67 | 33072.5 | 4.0 | 12_14_2_2 | 33071.7 | 0.7 |
| A68 | 33102.6 | 30.2 | 12_12_1_4 | 33101.7 | 0.8 |
| A69 | 33138.5 | 8.1 | 14_12_3_2 | 33135.7 | 2.8 |
| A70 | 33176.8 | 16.5 | 13_13_1_3 | 33175.8 | 1.0 |
| A71 | 33213.2 | 22.7 | 15_13_3_1 | 33207.8 | 5.5 |
| A72 | 33250.3 | 3.8 | 12_12_2_4 | 33247.8 | 2.5 |
| A73 | 33288.0 | 23.1 | 16_14_3_0 | 33283.8 | 4.1 |
| A74 | 33320.0 | 12.6 | 11_11_3_5 | 33319.8 | 0.2 |
| A75 | 33362.3 | 13.4 | 12_14_2_3 | 33362.8 | 0.5 |
| A76 | 33393.8 | 21.4 | 12_12_1_5 | 33392.8 | 1.0 |
| A77 | 33467.9 | 24.3 | 13_13_1_4 | 33466.8 | 1.0 |
| A78 | 33504.4 | 8.7 | 15_13_3_2 | 33500.9 | 3.5 |
| A79 | 33542.5 | 11.6 | 14_14_1_3 | 33540.9 | 1.6 |
| A80 | 33578.2 | 15.7 | 16_14_3_1 | 33574.9 | 3.3 |
| A81 | 33613.2 | 4.6 | 13_13_0_5 | 33611.9 | 1.3 |
| A82 | 33653.0 | 18.2 | 12_14_2_4 | 33653.9 | 0.9 |
| A83 | 33685.1 | 8.2 | 14_14_2_3 | 33686.9 | 1.9 |
| A84 | 33727.9 | 11.9 | 11_13_3_5 | 33726.0 | 1.9 |
| A85 | 33759.2 | 17.4 | 13_13_1_5 | 33757.9 | 1.3 |
| A86 | 33833.4 | 16.8 | 14_14_1_4 | 33832.0 | 1.4 |
| A87 | 33868.9 | 7.3 | 16_14_1_3 | 33865.0 | 3.9 |
| A88 | 33907.0 | 11.8 | 13_13_0_6 | 33903.0 | 4.0 |
| A89 | 33944.0 | 12.4 | 13_13_0_6 | 33945.0 | 1.0 |
| A90 | 34018.6 | 14.8 | 12_14_2_5 | 34018.0 | 0.6 |
| A91 | 34051.1 | 10.3 | 13_13_3_5 | 34050.1 | 1.0 |
| A92 | 34125.0 | 11.1 | 14_14_1_5 | 34123.1 | 2.0 |
| A93 | 34198.1 | 9.5 | 13_13_0_7 | 34194.1 | 4.0 |
| A94 | 34309.0 | 7.9 | 13_15_0_6 | 34309.1 | 0.2 |
| A95 | 34599.3 | 3.5 | 13_15_2_6 | 34601.3 | 2.0 |
| A96 | 34823.3 | 2.2 | 16_13_1_7 | 34826.3 | 3.0 |
| A97 | 34886.3 | 8.7 | 16_14_2_6 | 34884.3 | 2.0 |
Appendix 1—table 5
Summary of molecular weights (MWs) of B.1.351 receptor binding domain (RBD) glycoforms (Beta, Bx) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) |
|---|---|---|---|---|---|
| B1 | 31247.7 | 10.6 | 9_9_0_2 | 31255.0 | 7.3 |
| B2 | 31352.5 | 18.2 | 10_8_1_2 | 31360.0 | 7.6 |
| B3 | 31376.3 | 12.9 | 9_11_0_1 | 31370.0 | 6.2 |
| B4 | 31408.6 | 25.9 | 11_11_0_0 | 31403.1 | 5.5 |
| B5 | 31433.0 | 16.9 | 11_9_1_1 | 31434.0 | 1.0 |
| B6 | 31512.7 | 19.6 | 9_11_1_1 | 31516.1 | 3.4 |
| B7 | 31538.1 | 62.3 | 9_9_0_3 | 31546.1 | 8.0 |
| B8 | 31559.8 | 7.0 | 10_9_1_2 | 31563.1 | 3.2 |
| B9 | 31595.6 | 34.7 | 9_10_1_2 | 31604.1 | 8.6 |
| B10 | 31616.5 | 9.9 | 10_10_0_2 | 31620.1 | 3.6 |
| B11 | 31641.7 | 11.0 | 11_10_1_1 | 31637.1 | 4.6 |
| B12 | 31669.2 | 15.4 | 13_10_1_0 | 31670.1 | 0.9 |
| B13 | 31700.2 | 100.0 | 10_9_0_3 | 31708.1 | 7.9 |
| B14 | 31723.8 | 64.4 | 11_9_1_2 | 31725.1 | 1.3 |
| B15 | 31758.7 | 10.9 | 10_10_1_2 | 31766.2 | 7.5 |
| B16 | 31778.5 | 16.2 | 11_10_0_2 | 31782.2 | 3.7 |
| B17 | 31803.0 | 12.5 | 12_10_1_1 | 31799.2 | 3.8 |
| B18 | 31829.5 | 42.9 | 10_11_2_1 | 31824.2 | 5.3 |
| B19 | 31886.1 | 84.9 | 12_9_1_2 | 31887.2 | 1.1 |
| B20 | 31906.0 | 32.1 | 10_10_0_3 | 31911.2 | 5.2 |
| B21 | 31962.0 | 22.6 | 10_11_1_2 | 31969.3 | 7.2 |
| B22 | 31991.3 | 58.7 | 10_9_2_3 | 31000.2 | 8.9 |
| B23 | 32014.8 | 31.8 | 11_9_1_3 | 32016.2 | 1.5 |
| B24 | 32066.4 | 53.9 | 11_10_0_3 | 32073.3 | 6.9 |
| B25 | 32089.4 | 36.3 | 12_10_1_2 | 32090.3 | 0.9 |
| B26 | 32142.2 | 10.0 | 12_11_0_2 | 32147.3 | 5.1 |
| B27 | 32177.0 | 45.9 | 12_9_1_3 | 32178.3 | 1.3 |
| B28 | 32196.7 | 21.8 | 10_10_0_4 | 32202.3 | 5.6 |
| B29 | 32251.5 | 48.1 | 13_10_1_2 | 32252.3 | 0.9 |
| B30 | 32271.1 | 10.1 | 11_11_0_3 | 32276.3 | 5.3 |
| B31 | 32326.4 | 14.9 | 14_11_1_1 | 32326.4 | 0.1 |
| B32 | 32356.4 | 33.3 | 11_10_0_4 | 32364.4 | 8.0 |
| B33 | 32379.5 | 27.6 | 12_10_1_3 | 32381.4 | 1.9 |
| B34 | 32431.3 | 48.8 | 12_11_0_3 | 32438.4 | 7.1 |
| B35 | 32454.7 | 29.5 | 11_9_2_4 | 32453.4 | 1.3 |
| B36 | 32541.6 | 24.4 | 13_10_1_3 | 32543.4 | 1.8 |
| B37 | 32560.9 | 20.8 | 11_11_0_4 | 32567.4 | 6.6 |
| B38 | 32617.1 | 43.1 | 14_11_1_2 | 32617.5 | 0.4 |
| B39 | 32637.1 | 9.6 | 12_12_0_3 | 32641.5 | 4.4 |
| B40 | 32722.1 | 35.2 | 12_11_0_4 | 32729.5 | 7.4 |
| B41 | 32744.8 | 17.2 | 13_11_1_3 | 32746.5 | 1.7 |
| B42 | 32796.8 | 34.1 | 11_10_1_5 | 32801.5 | 4.7 |
| B43 | 32820.3 | 13.6 | 12_10_2_4 | 32818.5 | 1.8 |
| B44 | 32927.0 | 9.0 | 12_12_0_4 | 32932.6 | 5.6 |
| B45 | 32983.0 | 30.0 | 13_10_2_4 | 32980.6 | 2.4 |
| B46 | 33109.9 | 10.9 | 12_10_2_5 | 33109.6 | 0.3 |
| B47 | 33162.3 | 26.9 | 12_11_1_5 | 33166.6 | 4.4 |
| B48 | 33273.5 | 21.2 | 13_10_2_5 | 33271.7 | 1.8 |
| B49 | 33348.1 | 27.5 | 14_11_2_4 | 33345.7 | 2.4 |
| B50 | 33452.7 | 24.3 | 12_11_1_6 | 33457.7 | 5.1 |
| B51 | 33528.0 | 25.5 | 13_12_1_5 | 33531.8 | 3.8 |
| B52 | 33637.9 | 17.3 | 14_11_2_5 | 33636.8 | 1.1 |
| B53 | 33713.1 | 20.7 | 13_10_3_6 | 33708.8 | 4.3 |
| B54 | 33817.9 | 21.0 | 13_12_1_6 | 33822.9 | 5.0 |
| B55 | 33893.0 | 17.0 | 14_13_1_5 | 33896.9 | 3.9 |
| B56 | 34003.3 | 13.8 | 13_10_3_7 | 33999.9 | 3.4 |
| B57 | 34183.0 | 10.0 | 14_13_1_6 | 34188.0 | 5.0 |
| B58 | 34548.7 | 7.7 | 15_14_1_6 | 34553.1 | 4.5 |
| B59 | 34693.1 | 13.5 | 15_14_2_6 | 34699.2 | 6.1 |
Appendix 1—table 6
Summary of molecular weights (MWs) of B.1.351.2 receptor binding domain (RBD) glycoforms (Delta, Dx) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) |
|---|---|---|---|---|---|
| D1 | 31073.5 | 20.9 | 8_11_1_0 | 31079.0 | 5.5 |
| D2 | 31094.3 | 1.0 | 9_11_0_0 | 31095.0 | 0.7 |
| D3 | 31259.9 | 24.3 | 10_11_0_0 | 31257.1 | 2.8 |
| D4 | 31363.7 | 28.6 | 8_11_1_1 | 31370.1 | 6.4 |
| D5 | 31423.0 | 23.4 | 11_11_0_0 | 31419.1 | 3.9 |
| D6 | 31526.2 | 26.6 | 9_11_1_1 | 31532.2 | 5.9 |
| D7 | 31548.3 | 25.4 | 10_11_0_1 | 31548.2 | 0.2 |
| D8 | 31654.2 | 29.4 | 8_11_1_2 | 31661.2 | 7.0 |
| D9 | 31690.5 | 23.9 | 10_11_1_1 | 31694.2 | 3.7 |
| D10 | 31711.4 | 39.3 | 11_11_0_1 | 31710.2 | 1.2 |
| D11 | 31733.5 | 16.1 | 9_12_1_1 | 31735.2 | 1.7 |
| D12 | 31813.8 | 33.6 | 10_13_1_0 | 31809.3 | 4.5 |
| D13 | 31838.0 | 31.0 | 10_11_0_2 | 31839.3 | 1.3 |
| D14 | 31875.9 | 13.2 | 12_11_2_0 | 31873.3 | 2.6 |
| D15 | 31895.2 | 25.7 | 10_12_1_1 | 31897.3 | 2.1 |
| D16 | 31979.4 | 30.5 | 10_11_1_2 | 31985.3 | 5.9 |
| D17 | 31000.6 | 60.7 | 11_11_0_2 | 31001.3 | 0.8 |
| D18 | 32022.0 | 26.0 | 9_12_1_2 | 32026.3 | 4.3 |
| D19 | 32079.6 | 25.4 | 12_12_0_1 | 32075.3 | 4.2 |
| D20 | 32104.2 | 49.0 | 10_13_1_1 | 32100.4 | 3.8 |
| D21 | 32127.5 | 29.3 | 10_11_0_3 | 32130.3 | 2.8 |
| D22 | 32164.9 | 4.6 | 12_11_0_2 | 32163.4 | 1.6 |
| D23 | 32185.4 | 44.8 | 10_12_1_2 | 32188.4 | 3.0 |
| D24 | 32206.8 | 5.0 | 11_12_0_2 | 32204.4 | 2.4 |
| D25 | 32290.9 | 90.6 | 11_11_0_3 | 32292.4 | 1.5 |
| D26 | 32312.4 | 32.4 | 9_12_1_3 | 32317.4 | 5.1 |
| D27 | 32348.4 | 26.0 | 11_12_1_2 | 32350.4 | 2.1 |
| D28 | 32369.1 | 34.1 | 12_12_0_2 | 32366.4 | 2.7 |
| D29 | 32394.4 | 45.5 | 10_13_1_2 | 32391.5 | 3.0 |
| D30 | 32475.9 | 50.0 | 10_12_1_3 | 32479.5 | 3.5 |
| D31 | 32496.5 | 22.2 | 11_12_0_3 | 32495.5 | 1.0 |
| D32 | 32553.6 | 0.7 | 11_13_1_2 | 32553.5 | 0.1 |
| D33 | 32581.7 | 100.0 | 11_11_0_4 | 32583.5 | 1.8 |
| D34 | 32603.7 | 34.7 | 12_11_3_2 | 32601.5 | 2.2 |
| D35 | 32638.2 | 0.3 | 11_12_1_3 | 32641.5 | 3.3 |
| D36 | 32658.4 | 41.3 | 12_12_0_3 | 32657.5 | 0.8 |
| D37 | 32683.3 | 21.8 | 10_13_1_3 | 32682.6 | 0.7 |
| D38 | 32766.4 | 41.9 | 10_12_1_4 | 32770.6 | 4.2 |
| D39 | 32843.0 | 8.0 | 11_13_1_3 | 32844.6 | 1.7 |
| D40 | 32873.4 | 68.5 | 11_11_0_5 | 32874.6 | 1.2 |
| D41 | 32895.2 | 18.5 | 12_11_3_3 | 32892.6 | 2.6 |
| D42 | 32948.2 | 45.1 | 12_12_0_4 | 32948.6 | 0.4 |
| D43 | 33238.9 | 42.5 | 12_12_0_5 | 33239.7 | 0.8 |
| D44 | 33313.9 | 30.2 | 13_13_0_4 | 33313.8 | 0.1 |
| D45 | 33604.6 | 37.1 | 13_13_0_5 | 33604.9 | 0.2 |
| D46 | 33896.1 | 29.5 | 13_13_0_6 | 33896.0 | 0.2 |
| D47 | 33969.9 | 9.2 | 12_12_3_6 | 33969.0 | 0.9 |
| D48 | 34186.4 | 21.6 | 13_13_0_7 | 34187.0 | 0.7 |
| D49 | 34260.3 | 9.0 | 14_14_0_6 | 34261.1 | 0.7 |
Appendix 1—table 7
Summary of molecular weights (MWs) of B.1.1.529 receptor binding domain (RBD) glycoforms (Omicron, Ox) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) |
|---|---|---|---|---|---|
| O1 | 32489.2 | 40.2 | 10_11_1_3 | 32487.6 | 1.5 |
| O2 | 32538.4 | 45.7 | 11_9_3_3 | 32535.7 | 2.7 |
| O3 | 32559.4 | 5.6 | 11_12_1_2 | 32561.7 | 2.3 |
| O4 | 32576.0 | 23.0 | 10_10_1_4 | 32575.7 | 0.3 |
| O5 | 32595.1 | 74.6 | 11_10_2_3 | 32592.7 | 2.4 |
| O6 | 32615.4 | 31.4 | 12_10_1_3 | 32608.7 | 6.7 |
| O7 | 32651.1 | 29.0 | 11_11_1_3 | 32649.7 | 1.4 |
| O8 | 32670.2 | 24.9 | 12_11_2_2 | 32666.7 | 3.4 |
| O9 | 32702.3 | 42.5 | 11_12_0_3 | 32706.7 | 4.4 |
| O10 | 32723.8 | 87.1 | 10_10_2_4 | 32721.7 | 2.1 |
| O11 | 32744.0 | 2.5 | 11_10_3_3 | 32738.7 | 5.3 |
| O12 | 32761.0 | 11.3 | 12_10_2_3 | 32754.7 | 6.3 |
| O13 | 32779.8 | 53.6 | 10_11_1_4 | 32778.7 | 1.1 |
| O14 | 32798.7 | 23.0 | 11_11_2_3 | 32795.8 | 2.9 |
| O15 | 32829.1 | 62.1 | 11_9_3_4 | 32826.8 | 2.3 |
| O16 | 32852.5 | 27.6 | 11_12_1_3 | 32852.8 | 0.3 |
| O17 | 32885.7 | 85.7 | 11_10_2_4 | 32883.8 | 2.0 |
| O18 | 32905.7 | 29.7 | 12_10_3_3 | 32900.8 | 4.9 |
| O19 | 32942.2 | 12.1 | 11_11_1_4 | 32940.8 | 1.4 |
| O20 | 32960.8 | 40.8 | 12_11_2_3 | 32957.8 | 3.0 |
| O21 | 33014.9 | 100.0 | 12_12_1_3 | 33014.8 | 0.1 |
| O22 | 33035.6 | 25.5 | 11_10_3_4 | 33029.8 | 5.8 |
| O23 | 33070.2 | 52.1 | 12_13_0_3 | 33071.9 | 1.6 |
| O24 | 33089.8 | 52.7 | 11_11_2_4 | 33086.9 | 2.9 |
| O25 | 33120.4 | 52.4 | 13_11_2_3 | 33119.9 | 0.6 |
| O26 | 33144.7 | 28.3 | 11_12_1_4 | 33143.9 | 0.8 |
| O27 | 33177.5 | 50.7 | 11_10_2_5 | 33174.9 | 2.6 |
| O28 | 33195.9 | 33.2 | 12_10_3_4 | 33191.9 | 4.0 |
| O29 | 33217.4 | 13.2 | 12_13_1_3 | 33217.9 | 0.5 |
| O30 | 33233.9 | 1.6 | 11_11_1_5 | 33231.9 | 2.0 |
| O31 | 33251.6 | 58.4 | 12_11_2_4 | 33248.9 | 2.7 |
| O32 | 33271.2 | 15.4 | 13_11_3_3 | 33265.9 | 5.2 |
| O33 | 33306.1 | 83.2 | 12_12_1_4 | 33305.9 | 0.2 |
| O34 | 33326.7 | 28.9 | 13_12_2_3 | 33322.9 | 3.7 |
| O35 | 33361.5 | 30.3 | 12_13_0_4 | 33362.9 | 1.4 |
| O36 | 33380.8 | 61.4 | 13_13_1_3 | 33380.0 | 0.9 |
| O37 | 33402.5 | 0.3 | 14_13_0_3 | 33396.0 | 6.5 |
| O38 | 33435.9 | 27.5 | 11_12_1_5 | 33435.0 | 0.9 |
| O39 | 33454.8 | 15.1 | 12_12_2_4 | 33452.0 | 2.8 |
| O40 | 33486.2 | 24.5 | 14_12_0_4 | 33484.0 | 2.2 |
| O41 | 33508.3 | 18.3 | 12_13_1_4 | 33509.0 | 0.7 |
| O42 | 33542.1 | 37.0 | 12_11_2_5 | 33540.0 | 2.1 |
| O43 | 33561.5 | 24.8 | 15_13_0_3 | 33558.0 | 3.5 |
| O44 | 33597.5 | 25.2 | 12_12_1_5 | 33597.0 | 0.5 |
| O45 | 33617.3 | 33.1 | 13_12_2_4 | 33614.0 | 3.2 |
| O46 | 33671.8 | 47.2 | 13_13_1_4 | 33671.1 | 0.7 |
| O47 | 33691.7 | 16.4 | 14_13_0_4 | 33687.1 | 4.6 |
| O48 | 33726.3 | 13.7 | 13_14_0_4 | 33728.1 | 1.7 |
| O49 | 33745.7 | 35.3 | 12_12_2_5 | 33743.1 | 2.6 |
| O50 | 33776.2 | 16.1 | 14_12_2_4 | 33776.1 | 0.1 |
| O51 | 33800.4 | 17.0 | 12_13_1_5 | 33800.1 | 0.3 |
| O52 | 33833.8 | 3.0 | 14_13_1_4 | 33833.1 | 0.7 |
| O53 | 33851.7 | 28.4 | 15_13_0_4 | 33849.1 | 2.6 |
| O54 | 33874.5 | 10.3 | 13_14_1_4 | 33874.1 | 0.4 |
| O55 | 33907.8 | 21.7 | 13_12_2_5 | 33905.1 | 2.7 |
| O56 | 33927.2 | 14.4 | 14_12_3_4 | 33922.1 | 5.0 |
| O57 | 33963.1 | 27.1 | 13_13_1_5 | 33962.2 | 0.9 |
| O58 | 33982.3 | 13.7 | 14_13_0_5 | 33978.1 | 4.2 |
| O59 | 34037.0 | 32.6 | 14_14_1_4 | 34036.2 | 0.8 |
| O60 | 34058.2 | 3.7 | 13_12_3_5 | 34051.2 | 7.0 |
| O61 | 34090.9 | 8.8 | 12_13_1_6 | 34091.2 | 0.3 |
| O62 | 34110.9 | 11.6 | 13_13_2_5 | 34108.2 | 2.7 |
| O63 | 34142.7 | 22.5 | 15_13_0_5 | 34140.2 | 2.5 |
| O64 | 34165.6 | 5.1 | 13_14_1_5 | 34165.2 | 0.4 |
| O65 | 34199.3 | 6.6 | 15_14_1_4 | 34198.2 | 1.1 |
| O66 | 34217.1 | 20.9 | 14_12_1_6 | 34212.2 | 4.8 |
| O67 | 34274.9 | 20.8 | 14_13_2_5 | 34270.3 | 4.6 |
| O68 | 34327.5 | 26.7 | 14_14_1_5 | 34327.3 | 0.2 |
| O69 | 34346.6 | 15.5 | 13_12_3_6 | 34342.3 | 4.3 |
| O70 | 34384.5 | 0.9 | 13_10_4_7 | 34373.3 | 11.2 |
| O71 | 34402.4 | 23.3 | 13_13_2_6 | 34399.3 | 3.1 |
| O72 | 34457.1 | 9.0 | 13_14_1_6 | 34456.3 | 0.8 |
| O73 | 34506.7 | 18.8 | 14_12_1_7 | 34503.3 | 3.4 |
| O74 | 34529.7 | 3.1 | 12_13_4_6 | 34529.4 | 0.3 |
| O75 | 34564.3 | 25.0 | 14_13_0_7 | 34560.3 | 4.0 |
| O76 | 34620.0 | 26.6 | 14_14_1_6 | 34618.4 | 1.6 |
| O77 | 34638.4 | 23.0 | 15_14_2_5 | 34635.4 | 3.0 |
| O78 | 34658.8 | 12.7 | 16_14_3_4 | 34652.4 | 6.4 |
| O79 | 34768.4 | 0.9 | 14_14_2_6 | 34764.4 | 4.0 |
| O80 | 34798.6 | 29.8 | 14_12_1_8 | 34794.4 | 4.2 |
| O81 | 34840.4 | 0.5 | 13_13_3_7 | 34836.5 | 4.0 |
| O82 | 34858.9 | 20.4 | 14_13_0_8 | 34851.4 | 7.5 |
| O83 | 35019.8 | 14.5 | 15_13_0_8 | 35013.5 | 6.3 |
| O84 | 35041.2 | 10.5 | 13_14_3_7 | 35039.5 | 1.7 |
| O85 | 35220.7 | 23.8 | 15_14_0_8 | 35216.6 | 4.1 |
| O86 | 35312.3 | 2.6 | 15_13_2_8 | 35305.6 | 6.7 |
| O87 | 35354.0 | 19.8 | 15_14_1_8 | 35362.6 | 8.6 |
Appendix 1—table 8
Summary of molecular weights (MWs) of endoF3-treated WHu-Hu-1 receptor binding domain (RBD) glycoforms (eWTx) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) | Number of trimmed N-glycans |
|---|---|---|---|---|---|---|
| eWT1 | 28116.9 | 4.5 | 1_3_0_0 | 28123.9 | 7.0 | 2 |
| eWT2 | 28243.9 | 26.4 | 3_2_0_0 | 28244.9 | 1.0 | 2 |
| eWT3 | 28357.3 | 14.5 | 1_2_1_1 | 28358.0 | 0.6 | 2 |
| eWT4 | 28618.0 | 23.8 | 1_4_2_0 | 28619.1 | 1.0 | 2 |
| eWT5 | 28881.1 | 2.4 | 3_3_1_1 | 28885.1 | 4.0 | 2 |
| eWT6 | 29029.2 | 18.7 | 3_3_2_1 | 29031.2 | 2.1 | 2 |
| eWT7 | 29143.8 | 1.1 | 6_4_0_0 | 29137.2 | 6.5 | 1 |
| eWT8 | 29318.2 | 1.1 | 5_5_1_0 | 29324.3 | 6.1 | 1 |
| eWT9 | 29406.5 | 8.2 | 5_4_1_1 | 29412.3 | 5.8 | 1 |
| eWT10 | 29454.5 | 14.1 | 4_5_1_1 | 29453.4 | 1.2 | 1 |
| eWT11 | 29649.4 | 0.6 | 7_5_1_0 | 29648.4 | 1.0 | 1 |
| eWT12 | 29736.3 | 9.6 | 5_7_1_0 | 29730.5 | 5.8 | 1 |
| eWT13 | 29753.5 | 1.3 | 5_5_0_2 | 29760.5 | 6.9 | 1 |
| eWT14 | 30084.9 | 1.3 | 7_5_2_1 | 30085.6 | 0.7 | 1 |
| eWT15 | 30103.4 | 10.3 | 5_6_1_2 | 30109.6 | 6.2 | 1 |
| eWT16 | 31033.0 | 27.6 | 8_6_2_3 | 31032.9 | 0.1 | 1 |
| eWT17 | 31216.4 | 17.2 | 7_7_1_4 | 31219.0 | 2.5 | 1 |
| eWT18 | 31416.8 | 8.0 | 7_8_1_4 | 31422.0 | 5.2 | 1 |
| eWT19 | 31437.4 | 0.9 | 8_8_0_4 | 31438.0 | 0.7 | 1 |
| eWT20 | 31482.3 | 9.0 | 7_9_2_3 | 31480.1 | 2.2 | 1 |
| eWT21 | 31505.7 | 38.4 | 9_9_0_3 | 31512.1 | 6.4 | 0 |
| eWT22 | 31543.3 | 41.0 | 9_7_1_4 | 31543.1 | 0.2 | 1 |
| eWT23 | 31564.8 | 55.2 | 7_8_2_4 | 31568.1 | 3.3 | 1 |
| eWT24 | 31607.4 | 2.2 | 11_10_3_0 | 31604.1 | 3.2 | 0 |
| eWT25 | 31644.1 | 27.3 | 8_9_2_3 | 31642.1 | 2.0 | 1 |
| eWT26 | 31668.2 | 82.8 | 10_9_0_3 | 31674.1 | 5.9 | 0 |
| eWT27 | 31691.0 | 33.8 | 11_9_1_2 | 31691.1 | 0.2 | 0 |
| eWT28 | 31708.4 | 9.9 | 9_10_0_3 | 31715.2 | 6.7 | 0 |
| eWT29 | 31727.6 | 58.6 | 10_10_1_2 | 31732.2 | 4.5 | 0 |
| eWT30 | 31748.3 | 42.7 | 11_10_0_2 | 31748.2 | 0.1 | 0 |
| eWT31 | 31770.9 | 29.2 | 12_10_3_0 | 31766.2 | 4.7 | 0 |
| eWT32 | 31795.9 | 25.7 | 10_11_0_2 | 31789.2 | 6.7 | 0 |
| eWT33 | 31833.6 | 35.0 | 11_9_2_2 | 31837.2 | 3.6 | 0 |
| eWT34 | 31854.4 | 95.9 | 9_10_1_3 | 31861.2 | 6.8 | 0 |
| eWT35 | 31872.6 | 14.1 | 10_10_2_2 | 31878.2 | 5.6 | 0 |
| eWT36 | 31893.5 | 1.5 | 11_10_1_2 | 31894.2 | 0.7 | 0 |
| eWT37 | 31912.9 | 51.6 | 12_10_0_2 | 31910.2 | 2.6 | 0 |
| eWT38 | 31933.2 | 26.1 | 10_11_1_2 | 31935.3 | 2.0 | 0 |
| eWT39 | 31958.1 | 100.0 | 11_11_0_2 | 31951.2 | 6.9 | 0 |
| eWT40 | 31980.4 | 28.5 | 13_11_0_1 | 31984.3 | 3.9 | 0 |
| eWT41 | 32017.7 | 36.8 | 10_10_1_3 | 32023.3 | 5.5 | 0 |
| eWT42 | 32060.9 | 6.2 | 12_10_1_2 | 32056.3 | 4.6 | 0 |
Appendix 1—table 9
Summary of molecular weights (MWs) of endoF3-treated B.1.351.2 receptor binding domain (RBD) glycoforms (eDx) with respective relative abundances identified by ESI-MS and putative H_N_F_S combinations.
| RBD | MeasuredMW (Da) | Relative abundance | H_N_F_S combination | Theoretical MW (Da) | Mass difference (Da) | Number of trimmed N-glycans |
|---|---|---|---|---|---|---|
| eD1 | 28681.8 | 2.2 | 4_3_0_0 | 28680.1 | 1.7 | 2 |
| eD2 | 28720.3 | 24.9 | 3_4_0_0 | 28721.1 | 0.8 | 2 |
| eD3 | 28741.8 | 35.3 | 2_2_2_1 | 28736.1 | 5.6 | 2 |
| eD4 | 28762.6 | 9.4 | 2_5_0_0 | 28762.2 | 0.5 | 2 |
| eD5 | 28782.3 | 33.5 | 1_3_2_1 | 28777.2 | 5.2 | 2 |
| eD6 | 28803.6 | 25.9 | 3_3_2_0 | 28810.2 | 6.5 | 2 |
| eD7 | 28826.2 | 19.8 | 4_3_1_0 | 28826.2 | 0.1 | 2 |
| eD8 | 28867.3 | 82.4 | 3_4_1_0 | 28867.2 | 0.1 | 2 |
| eD9 | 28888.2 | 52.6 | 4_4_0_0 | 28883.2 | 5.0 | 2 |
| eD10 | 28907.6 | 20.6 | 3_2_2_1 | 28898.2 | 9.4 | 2 |
| eD11 | 28927.8 | 100.0 | 3_5_0_0 | 28924.2 | 3.6 | 2 |
| eD12 | 28949.2 | 57.5 | 3_3_1_1 | 28955.2 | 6.0 | 2 |
| eD13 | 28970.6 | 38.3 | 4_3_0_1 | 28971.2 | 0.6 | 2 |
| eD14 | 28990.7 | 30.3 | 2_4_1_1 | 28996.2 | 5.5 | 2 |
| eD15 | 29011.6 | 32.3 | 3_4_0_1 | 29012.2 | 0.6 | 2 |
| eD16 | 29032.7 | 24.1 | 4_4_1_0 | 29029.2 | 3.5 | 2 |
| eD17 | 29053.6 | 52.1 | 3_2_1_2 | 29043.2 | 10.4 | 2 |
| eD18 | 29074.7 | 51.9 | 3_5_1_0 | 29070.3 | 4.4 | 2 |
| eD19 | 29095.1 | 15.7 | 3_3_2_1 | 29101.3 | 6.2 | 2 |
| eD20 | 29114.2 | 37.7 | 4_3_1_1 | 29117.3 | 3.1 | 2 |
| eD21 | 29135.1 | 26.0 | 2_4_0_2 | 29141.3 | 6.2 | 2 |
| eD22 | 29158.3 | 34.9 | 3_4_1_1 | 29158.3 | 0.0 | 2 |
| eD23 | 29179.7 | 25.3 | 4_4_2_0 | 29175.3 | 4.4 | 2 |
| eD24 | 29198.8 | 17.6 | 3_2_2_2 | 29189.3 | 9.6 | 2 |
| eD25 | 29219.2 | 43.7 | 3_5_0_1 | 29215.3 | 3.9 | 2 |
| eD26 | 29240.2 | 24.3 | 3_3_1_2 | 29246.3 | 6.1 | 2 |
| eD27 | 29261.4 | 16.4 | 4_3_2_1 | 29263.3 | 1.9 | 2 |
| eD28 | 29281.5 | 6.4 | 2_4_1_2 | 29287.3 | 5.9 | 2 |
| eD29 | 29301.3 | 1.9 | 3_4_2_1 | 29304.3 | 3.0 | 2 |
| eD30 | 29302.0 | 1.2 | 3_4_0_2 | 29303.3 | 1.3 | 2 |
| eD31 | 29344.9 | 24.8 | 5_4_2_0 | 29337.4 | 7.6 | 2 |
| eD32 | 29366.8 | 5.0 | 3_5_1_1 | 29361.4 | 5.5 | 2 |
| eD33 | 29405.7 | 10.1 | 4_3_1_2 | 29408.4 | 2.7 | 2 |
| eD34 | 29427.1 | 3.8 | 2_4_2_2 | 29433.4 | 6.3 | 2 |
Appendix 1—table 10
Glycopeptide analysis of wild-type (WT) receptor binding domain (RBD).
| N-site | Peptide sequence | N-glycan | Retention time (min) | Measured m/z | Charge state | Mass accuracy (ppm) | MS area |
|---|---|---|---|---|---|---|---|
| N331 | PNITNLCPFGEV | A2S2 | 18.0 | 1170.471 | 3 | –3.66 | 88472 |
| N331 | PNITNLCPFGEV | A2S2 | 18.0 | 1755.705 | 2 | –2.48 | 70771 |
| N331 | PNITNLCPFGEV | A1G1F | 18.2 | 1354.577 | 2 | 0.45 | 157724 |
| N331 | PNITNLCPFGEV | A1G0F | 18.3 | 1273.551 | 2 | 0.14 | 85745 |
| N331 | PNITNLCPFGEV | A1S1F | 18.6 | 1500.124 | 2 | 0.04 | 65793 |
| N331 | FPNIT | A2G2B | 27.8 | 1209.489 | 2 | –2.73 | 57370 |
| N343 | GEVFNATR | A2S1G1FB | 31.0 | 1579.132 | 2 | –3.43 | 44341 |
| N343 | NATRF | A2G2F | 6.3 | 1189.484 | 2 | 0.33 | 107145 |
| N343 | NATRF | A2G1F | 6.5 | 1107.955 | 2 | 0.30 | 46566 |
| N343 | NATRF | A2S1G1F | 7.0 | 1335.032 | 2 | 0.75 | 38217 |
| N343 | PFGEVFNATR | A2G2 | 13.5 | 1381.076 | 2 | –3.29 | 272764 |
| N343 | PFGEVFNATR | A2G1 | 13.6 | 1300.049 | 2 | –3.69 | 150239 |
| N343 | PFGEVFNATR | A2S1G1 | 13.7 | 1526.623 | 2 | –2.90 | 305875 |
| N343 | PFGEVFNATR | A2S1G0 | 13.8 | 1445.597 | 2 | –3.53 | 164686 |
Appendix 1—table 11
Glycopeptide analysis of endoF3-treated wild-type (WT) receptor binding domain (RBD).
| N-site | Peptide sequence | N-glycan | Retention time (min) | Measured m/z | Charge state | Mass accuracy (ppm) | MS area |
|---|---|---|---|---|---|---|---|
| N331 | PNITNLCPFGEVF | A1G0F | 18.9 | 1347.585 | 2 | –3.88 | 21591 |
| N331 | PNITNLCPFGE | A2G0FB | 19.5 | 1427.087 | 2 | –5.81 | 83634 |
| N331 | PNITNLCPFGEV | A1G1F | 19.6 | 1354.575 | 2 | –1.09 | 112872 |
| N331 | PNITNLCPFGEV | A1G0F | 19.7 | 1273.551 | 2 | –1.21 | 52498 |
| N331 | PNITNLCPFGEV | A1S1F | 19.9 | 1500.122 | 2 | –1.51 | 59378 |
| N331 | PNITNLCPF | A2G1 | 26.2 | 827.350 | 3 | 1.33 | 49111 |
| N331 | PNITNLCPF | A2G1 | 26.9 | 1240.524 | 2 | 2.81 | 25854 |
| N331 | PNITNL | A1G1 | 28.6 | 964.915 | 2 | 0.19 | 19156 |
| N343 | NATRF | A2G2F | 6.3 | 1189.483 | 2 | 0.54 | 32460 |
| N343 | NATRF | A2G1F | 6.4 | 1107.955 | 2 | 0.19 | 47037 |
| N343 | NATRF | A2G0F | 6.5 | 1027.430 | 2 | 0.50 | 23831 |
| N343 | NATRF | A2S1G1F | 7.5 | 1335.032 | 2 | 0.75 | 34621 |
| N343 | NATRF | A2S2F | 7.8 | 1480.580 | 2 | 1.09 | 24974 |
| N343 | NATRF | GnF | 8.2 | 957.453 | 1 | 0.28 | 17273 |
| N343 | NATRF | GnF | 8.2 | 479.230 | 2 | 0.53 | 255747 |
| N343 | NATR | A2G0 | 18.0 | 880.363 | 2 | –2.3 | 34690 |
| N343 | NATRFASVY | A2S1G1 | 9.4 | 736.560 | 4 | 4.72 | 103643 |
| N343 | GEVFNATR | GnF | 11.3 | 621.797 | 2 | 0.83 | 14586 |
| N343 | GEVFNATR | GnF | 15.5 | 414.868 | 3 | 3.39 | 15706 |
| N343 | GEVFNATR | GnF | 15.7 | 414.868 | 3 | 3.78 | 18198 |
| N343 | GEVFNATRF | GnF | 17.6 | 695.331 | 2 | 1.23 | 17392 |
| N343 | GEVFNATRF | A2G0F | 23.2 | 1243.537 | 2 | 4.76 | 17779 |
