Site-specific analysis of the SARS-CoV-2 glycan shield

Yasunori Watanabe; Joel D. Allen; Daniel Wrapp; Jason S. McLellan; Max Crispin

Three 30 μg aliquots of SARS-CoV-2 S protein were denatured for 1h in 50 mM Tris/HCl, pH 8.0 containing 6 M of urea and 5 mM dithiothreitol (DTT). Next, the S protein were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubated for 1h in the dark, . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.26.010322 doi: bioRxiv preprint followed by a 1h incubation with 20 mM DTT to eliminate residual IAA. The alkylated Env proteins were buffer-exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (3 kDa) and digested separately overnight using trypsin chymotrypsin or alpha lytic protease (Mass Spectrometry Grade, Promega) at a ratio of 1:30 (w/w). The next day, the peptides were dried and extracted using C18 Zip-tip (MerckMilipore). The peptides were dried again, re-suspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Easy-nLC 1200 (Thermo Fisher Glycopeptide fragmentation data were extracted from the raw file using Byonic TM (Version 3.5) and Byologic TM software (Version 3.5; Protein Metrics Inc.). The glycopeptide fragmentation data were evaluated manually for each glycopeptide; the peptide was scored as true-positive when the correct b and y fragment ions were observed along with oxonium ions corresponding to the glycan identified. The MS data was searched using the Protein Metrics 305 N-glycan library. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence. All charge states for a single glycopeptide were summed. The precursor mass tolerance was set at 4 ppm and 10 ppm for fragments. A 1% false discovery rate (FDR) was applied. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected. HexNAc(2)Hex(9−5) was classified as M9 to M5. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.26.010322 doi: bioRxiv preprint and fucosylation. If all of the following compositions have a fucose they are assigned into the FA categories. HexNAc(3)Hex(3-4)X is assigned as A1, HexNAc(4)X is A2/A1B, HexNAc(5)X is A3/A2B, and HexNAc(6)X is A4/A3B. As this fragmentation method does not provide linkage information compositional isomers are group, so for example a triantennary glycan contains HexNAc 5 but so does a biantennary glycans with a bisect. Any glycan containing at least one sialic acid was counted as sialylated.