Analysis of N-linked and O-linked Glycans Released from SARS-Cov-2 S and Human ACE2 Proteins
Aliquots of approximately 25-50 μg of S or ACE2 protein were processed for glycan analysis as previously described (Aoki et al., 2007; Aoki et al., 2008). For N-linked glycan analysis, the proteins were digested with trypsin. Following trypsinization, glycopeptides were enriched by C18 Sep-Pak and subjected to PNGaseF digestion to release N-linked glycans. Following PNGaseF digestion, released glycans were separated from residual glycosylated peptides bearing O-linked glycans by C18 Sep-Pak. O-glycosylated peptides were eluted from the Sep-Pak and subjected to reductive β-elimination to release the O-glycans. Another 25-50 μg aliquot of each protein was denatured with SDS and digested with PNGaseF to remove N-linked glycans. The de-N-glycosylated, intact protein was precipitated with cold ethanol and then subjected to reductive β-elimination to release O-glycans. The profiles of O-glycans released from peptides or from intact protein were found to be comparable. N- and O-linked glycans released from glycoproteins were permethylated with methyliodide according to the method of Anumula and Taylor prior to MS analysis (Anumula and Taylor, 1992). Glycan structural analysis was performed using an LTQ-Orbitrap instrument (Orbitrap Discovery, Thermo Fisher Scientific). Detection and relative quantification of the prevalence of individual glycans was accomplished using the total ion mapping (TIM) and neutral loss scan (NL scan) functionality of the Xcalibur software package version 2.0 (Thermo Fisher Scientific) as previously described (Aoki et al., 2007; Aoki et al., 2008). Mass accuracy and detector response was tuned with a permethylated oligosaccharide standard in positive ion mode. For fragmentation by collision-induced dissociation (CID in MS2 and MSn), normalized collision energy of 45% was applied. Most permethylated glycans were identified as singly or doubly charged, sodiated species in positive mode. Sulfated N-glycans were detected as singly or doubly charged, deprotonated species in negative ion mode. Peaks for all charge states were deconvoluted by the charge state and summed for quantification. All spectra were manually interpreted and annotated. The explicit identities of individual monosaccharide residues have been assigned based on known human biosynthetic pathways. Graphical representations of monosaccharide residues are consistent with the Symbol Nomenclature for Glycans (SNFG), which has been broadly adopted by the glycomics community (Varki et al., 2015). The MS-based glycomics data generated in these analyses and the associated annotations are presented in accordance with the MIRAGE standards and the Athens Guidelines (Wells et al., 2013). Data annotation and assignment of glycan accession identifiers were facilitated by GRITS Toolbox, GlyTouCan, GNOme, and GlyGen (Kahsay et al., 2020; Tiemeyer et al., 2017; Weatherly et al., 2019; York et al., 2020).