Method Details

Expression, Purification, and Characterization of SARS-CoV-2 S and Human ACE2 Proteins
To express a stabilized ectodomain of Spike protein, a synthetic gene encoding residues 1−1208 of SARS-CoV-2 Spike with the furin cleavage site (residues 682–685) replaced by a “GGSG” sequence, proline substitutions at residues 986 and 987, and a foldon trimerization motif followed by a C-terminal 6xHisTag was created and cloned into the mammalian expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg, MD). The expression construct was transiently transfected in HEK293F cells using polyethylenimine (Polysciences, Inc, Warrington, PA). Protein was purified from cell supernatants using Ni-NTA resin (QIAGEN, Germany), the eluted fractions containing S protein were pooled, concentrated, and further purified by gel filtration chromatography on a Superose 6 column (GE Healthcare). Negative stain electron microscopy (EM) analysis was performed as described (Shaik et al., 2019). Briefly, analysis was performed at room temperature with a magnification of 52,000x and a defocus value of 1.5 μm following low-dose procedures, using a Philips Tecnai F20 electron microscope (Thermo Fisher Scientific) equipped with a Gatan US4000 CCD camera and operated at voltage of 200 kV. The DNA fragment encoding human ACE2 (1-615) with a 6xHis tag at C terminus was synthesized by Genscript and cloned to the vector pCMV-IRES-puro. The expression construct was transfected in HEK293F cells using polyethylenimine. The medium was discarded and replaced with FreeStyle 293 medium after 6-8 h. After incubation in 37°C with 5.5% CO2 for 5 days, the supernatant was collected and loaded to Ni-NTA resin for purification. The elution was concentrated and further purified by a Superdex 200 column.

In-Gel Analysis of SARS-CoV-2 S and Human ACE2 Proteins
A 3.5-μg aliquot of SARS-CoV-2 S protein as well as a 2-μg aliquot of human ACE2 were combined with Laemmli sample buffer, analyzed on a 4%–12% Invitrogen NuPage Bis-Tris gel using the MES pH 6.5 running buffer, and stained with Coomassie Brilliant Blue G-250.

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).

Analysis of Disulfide Bonds for SARS-Cov-2 S Protein by LC-MS
Two 10-μg aliquots of SARS-CoV-2 S protein were denatured by incubating with 20% acetonitrile at room temperature and alkylated by 13.75 mM of iodoacetamide at room temperature in dark. The two aliquots of proteins were then digested respectively using alpha lytic protease, or a combination of trypsin, Lys-C and Glu-C. Following digestion, the proteins were deglycosylated by PNGaseF treatment. The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min. The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C. Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following electron transfer dissociation (ETD) were collected in the Orbitrap at 15k resolution. The raw spectra were analyzed by Byonic (v3.8.13, Protein Metrics Inc.) with mass tolerance set as 20 ppm for both precursors and fragments. The search output was filtered at 1% false discovery rate and 10 ppm mass error. The spectra assigned as cross-linked peptides were manually evaluated for Cys0015.

Analysis of Site-Specific N-linked Glycopeptides for SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
Four 3.5-μg aliquots of SARS-CoV-2 S protein were reduced by incubating with 10 mM of dithiothreitol at 56°C and alkylated by 27.5 mM of iodoacetamide at room temperature in dark. The four aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, a combination of trypsin and Glu-C, or a combination of Glu-C and AspN. Three 10-μg aliquots of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark. The three aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, or a combination of trypsin and Lys-C. The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min. The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C. Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher-energy collisional dissociation (HCD) with stepped collision energy (15%, 25%, 35%) were collected in the Orbitrap at 15k resolution. pGlyco v2.2.2 (Liu et al., 2017) was used for database searches with mass tolerance set as 20 ppm for both precursors and fragments. The database search output was filtered to reach a 1% false discovery rate for glycans and 10% for peptides. Quantitation was performed by calculating spectral counts for each glycan composition at each site. Any N-linked glycan compositions identified by only one spectra were removed from quantitation. N-linked glycan compositions were categorized into 22 classes (including Unoccupied): HexNAc(2)Hex(9∼5)Fuc(0∼1) was classified as M9 to M5 respectively; HexNAc(2)Hex(4∼1)Fuc(0∼1) was classified as M1-M4; HexNAc(3∼6)Hex(5∼9)Fuc(0)NeuAc(0∼1) was classified as Hybrid with HexNAc(3∼6)Hex(5∼9)Fuc(1∼2)NeuAc(0∼1) classified as F-Hybrid; Complex-type glycans are classified based on the number of antenna, fucosylation, and sulfation: HexNAc(3)Hex(3∼4)Fuc(0)NeuAc(0∼1) is assigned as A1 with HexNAc(3)Hex(3∼4)Fuc(1∼2)NeuAc(0∼1) assigned as F-A1; HexNAc(4)Hex(3∼5)Fuc(0)NeuAc(0∼2) is assigned as A2/A1B with HexNAc(4)Hex(3∼5)Fuc(1∼5)NeuAc(0∼2) assigned as F-A2/A1B; HexNAc(5)Hex(3∼6)Fuc(0)NeuAc(0∼3) is assigned as A3/A2B with HexNAc(5)Hex(3∼6)Fuc(1∼3)NeuAc(0∼3) assigned as F-A3/A2B; HexNAc(6)Hex(3∼7)Fuc(0)NeuAc(0∼4) is assigned as A4/A3B with HexNAc(6)Hex(3∼7)Fuc(1∼3)NeuAc(0∼4) assigned as F-A4/A3B; HexNAc(7)Hex(3∼8)Fuc(0)NeuAc(0∼1) is assigned as A5/A4B with HexNAc(7)Hex(3∼8)Fuc(1∼3)NeuAc(0∼1) as F-A5/A4B; HexNAc(8)Hex(3∼9)Fuc(0) is assigned as A6/A5B with HexNAc(8)Hex(3∼9)Fuc(1) assigned as F-A6/A5B; any glycans identified with a sulfate are assigned as Sulfated.

Analysis of Deglycosylated SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
Three 3.5-μg aliquots of SARS-CoV-2 S protein were reduced by incubating with 10 mM of dithiothreitol at 56°C and alkylated by 27.5 mM of iodoacetamide at room temperature in dark. The three aliquots were then digested respectively using chymotrypsin, Asp-N, or a combination of trypsin and Glu-C. Two 10-μg aliquots of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark. The two aliquots were then digested respectively using chymotrypsin, or a combination of trypsin and Lys-C. Following digestion, the proteins were deglycosylated by Endoglycosidase H followed by PNGaseF treatment in the presence of 18O water. The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min. The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C. Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following collision-induced dissociation (CID) at 38% collision energy were collected in the ion trap. The spectra were analyzed using SEQUEST (Proteome Discoverer 1.4) with mass tolerance set as 20 ppm for precursors and 0.5 Da for fragments. The search output was filtered using ProteoIQ (v2.7) to reach a 1% false discovery rate at protein level and 10% at peptide level. Occupancy of each N-linked glycosylation site was calculated using spectral counts assigned to the 18O-Asp-containing (PNGaseF-cleaved) and/or HexNAc-modified (EndoH-cleaved) peptides and their unmodified counterparts.

Analysis of Site-Specific O-linked Glycopeptides for SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
Three 10-μg aliquots of SARS-CoV-2 S protein and one 10-μg aliquot of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark. The four aliquots were then digested respectively using trypsin, Lys-C, Arg-C, or a combination of trypsin and Lys-C. Following digestion, the proteins were deglycosylated by PNGaseF treatment and then digested with O-protease OpeRATOR®. The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min. The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid. The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C. Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher-energy collisional dissociation (HCD) with stepped collision energy (15%, 25%, 35%) or electron transfer dissociation (ETD) were collected in the Orbitrap at 15k resolution. The raw spectra were analyzed by Byonic (v3.8.13) with mass tolerance set as 20 ppm for both precursors and fragments. MS/MS filtering was applied to only allow for spectra where the oxonium ions of HexNAc were observed. The search output was filtered at 1% false discovery rate and 10 ppm mass error. The spectra assigned as O-linked glycopeptides were manually evaluated. Quantitation was performed by calculating spectral counts for each glycan composition at each site. Any O-linked glycan compositions identified by only one spectra were removed from quantitation. Occupancy of each O-linked glycosylation site was calculated using spectral counts assigned to any glycosylated peptides and their unmodified counterparts from searches without MS/MS filtering.

Sequence Analysis of SARS-CoV-2 S and Human ACE2 Proteins
The genomes of SARS-CoV as well as bat and pangolin coronavirus sequences reported to be closely related to SARS-CoV-2 were downloaded from NCBI. The S protein sequences from all of those genomes were aligned using EMBOSS needle v6.6.0 (Rice et al., 2000) via the EMBL-EBI provided web service (Madeira et al., 2019). Manual analysis was performed in the regions containing canonical N-glycosylation sequons (N-X-S/T). For further sequence analysis of SARS-CoV-2 S variants, the genomes of SARS-CoV-2 were downloaded from NCBI and GISAID and further processed using Biopython 1.76 to extract all sequences annotated as “surface glycoprotein” and to remove any incomplete sequence as well as any sequence containing unassigned amino acids. For sequence analysis of human ACE2 variants, the single nucleotide polymorphisms (SNPs) of ACE2 were extracted from the NCBI dbSNP database and filtered for missense mutation entries with a reported minor allele frequency. Manual analysis was performed on both SARS-CoV-2 S and human ACE2 variants to further examine the regions containing canonical N-glycosylation sequons (N-X-S/T). LibreOffice Writer and its macro capabilities was used to shade regions on the linear sequence of S and ACE2.

3D Structural Modeling and Molecular Dynamics Simulation of Glycosylated SARS-CoV-2 S and Human ACE2 Proteins
SARS-CoV-2 Spike (S) protein structure and ACE2 co-complex – A 3D structure of the prefusion form of the S protein (RefSeq: YP_009724390.1, UniProt: P0DTC2 SPIKE_SARS2), based on a Cryo-EM structure (PDB code 6VSB) (Wrapp et al., 2020), was obtained from the SWISS-MODEL server (swissmodel.expasy.org). The model has 95% coverage (residues 27 to 1146) of the S protein. The receptor binding domain (RBD) in the “open” conformation was replaced with the RBD from an ACE2 co-complex (PDB code 6M0J) by grafting residues C336 to V524.
Glycoform generation – Glycans (detected by glycomics) were selected for installation on glycosylated S and ACE2 sequons (detected by glycoproteomics) based on three sets of criteria designed to reasonably capture different aspects of glycosylation microheterogeneity. We denote the first of these glycoform models as “Abundance.” The glycans selected for installation to generate the Abundance model were chosen because they were identified as the most abundant glycan structure (detected by glycomics) that matched the most abundant glycan composition (detected by glycoproteomics) at each individual site. We denote the second glycoform model as “Oxford Class.” The glycans selected for installation to generate the Oxford Class model were chosen because they were the most abundant glycan structure, (detected by glycomics) that was contained within the most highly represented Oxford classification group (detected by glycoproteomics) at each individual site (Figure S7; Tables S1 and S8). Finally, we denote the third glycoform model as “Processed.” The glycans selected for installation to generate the Processed model were chosen because they were the most highly trimmed, elaborated, or terminally decorated structure (detected by glycomics) that corresponded to a composition (detected by glycoproteomics) which was present at ≥ 1/3rd of the abundance of the most highly represented composition at each site (Table S1). 3D structures of the three glycoforms (Abundance, Oxford Class, Processed) were generated for the SARS-CoV-2 S protein alone, and in complex with the glycosylated ACE2 protein. The glycoprotein builder available at GLYCAM-Web (www.glycam.org) was employed together with an in-house program that adjusts the asparagine side chain torsion angles and glycosidic linkages within known low-energy ranges (Nivedha et al., 2014) to relieve any atomic overlaps with the core protein, as described previously (Grant et al., 2016; Peng et al., 2017).
Energy minimization and Molecular dynamics (MD) simulations – Each glycosylated structure was placed in a periodic box of TIP3P water molecules with a 10 Å buffer between the solute and the box edge. Energy minimization of all atoms was performed for 20,000 steps (10,000 steepest decent, followed by 10,000 conjugant gradient) under constant pressure (1 atm) and temperature (300 K) conditions. All MD simulations were performed under nPT conditions with the CUDA implementation of the PMEMD (Götz et al., 2012; Salomon-Ferrer et al., 2013) simulation code, as present in the Amber14 software suite (University of California, San Diego). The GLYCAM06j force field (Kirschner et al., 2008) and Amber14SB force field (Maier et al., 2015) were employed for the carbohydrate and protein moieties, respectively. A Berendsen barostat with a time constant of 1 ps was employed for pressure regulation, while a Langevin thermostat with a collision frequency of 2 ps-1 was employed for temperature regulation. A nonbonded interaction cut-off of 8 Å was employed. Long-range electrostatics were treated with the particle-mesh Ewald (PME) method (Darden and Pedersen, 1993). Covalent bonds involving hydrogen were constrained with the SHAKE algorithm, allowing an integration time step of 2 fs to be employed. The energy minimized coordinates were equilibrated at 300K over 400 ps with restraints on the solute heavy atoms. Each system was then equilibrated with restraints on the Ca atoms of the protein for 1ns, prior to initiating 4 independent 250 ns production MD simulations with random starting seeds for a total time of 1 μs per system, with no restraints applied.
Antigenic surface analysis. A series of 3D structure snapshots of the simulation were taken at 1 ns intervals and analyzed in terms of their ability to interact with a spherical probe based on the average size of hypervariable loops present in an antibody complementarity determining region (CDR), as described recently (https://www.biorxiv.org/content/10.1101/2020.04.07.030445v2). The percentage of simulation time each residue was exposed to the AbASA probe was calculated and plotted onto both the 3D structure and primary sequence.

Analysis of SARS-CoV-2 Spike VSV Pseudoparticles (ppVSV-SARS-2-S)
293T cells were transfected with an expression plasmid encoding SARS-CoV-2 Spike (pcDNAintron-SARS-2-SΔ19). To increase cell surface expression, the last 19 amino acids containing the Golgi retention signal were removed. Two SΔ19 constructs were compared, one started with Met1 and the other with Met2. Twenty-four h following transfection, cells were transduced with ppVSVΔG-VSV-G (particles that were pseudotyped with VSV-G in trans). One h following transduction cells were extensively washed and media was replaced. Supernatant containing particles were collected 12-24 h following transduction and cleared through centrifugation. Cleared supernatant was frozen at −80°C for future use. Target cells Vero E6 were seeded in 24-well plates (5x105 cells/mL) at a density of 80% coverage. The following day, ppVSV-SARS-2-S/GFP particles were transduced into target cells for 60 min, particles pseudotyped with VSV-G, Lassa virus GP, or no glycoprotein were included as controls. 24 h following transduction, transduced cells were released from the plate with trypsin, fixed with 4% formaldehyde, and GFP-positive virus-transduced cells were quantified using flow cytometry (Bectin Dickson BD-LSRII). To quantify the ability of various SARS-CoV-2 S mutants to mediate fusion, effector cells (HEK293T) were transiently transfected with the indicated pcDNAintron-SARS-2-S expression vector or measles virus H and F (Brindley et al., 2014). Effector cells were infected with MVA-T7 four h following transduction to produce the T7 polymerase (Paal et al., 2009). Target cells naturally expressing the receptor ACE2 (Vero) or ACE2 negative cells (HEK293T) were transfected with pTM1-luciferase, which encodes for firefly luciferase under the control of a T7 promoter (Brindley and Plemper, 2010). 24 h following transfection, the target cells were lifted and added to the effector cells at a 1:1 ratio. 4 h following co-cultivation, cells were washed, lysed and luciferase levels were quantified using Promega’s Steady-Glo substrate. To visualize cell-to-cell fusion, Vero cells were co-transfected with pGFP and the pcDNAintron-SARS-2-S constructs. 24 h following transfection, syncytia was visualized by fluorescence microscopy.