Protein Identification by Mass Spectrometry
All materials were supplied by ThermoFisher unless otherwise stated. Briefly, protein samples in 50 mM ammonium bicarbonate (ABC), 5 mM tris(2-carboxyethyl)phosphine-HCl were reduced by incubation at 37°C for 30 min. S-alkylation was performed by the addition of 1 µL 100 mM methyl methanethiosulfonate in isopropanol. For proteolytic digestion, 1.5 µL 0.2% ProteaseMax surfactant in 50 mM ABC and 2 µL 0.2g/L trypsin/endoproteinase Lys-C mixture (Promega) were added followed by incubation at 37°C for 16 h. Proteolysis was stopped and the surfactant hydrolyzed by the addition of 0.5% trifluoroacetic acid (TFA). The samples were desalted using HyperSep Hypercarb solid phase extraction tips and dried by vacuum centrifugation. For RPLC-MS, samples in 0.5% TFA, 3% acetonitrile (ACN) were injected. Peptides were separated using an RSLCnano system with a PepSwift PS-DVB monolithic column using a gradient from 97% solvent A (0.1% formic acid) to 35% solvent B (0.1% formic acid, 80% ACN). Mass spectra were acquired on a Q Exactive HF quadrupole-Orbitrap instrument, with automated data dependent switching between full-MS and tandem MS/MS scans. Proteins were identified by converting the MS data into Mascot Generic Format (MGF) files and analyzed against human and Chinese hamster reference proteome databases with the spike glycoprotein construct sequence inserted (www.uniprot.org) using Mascot Daemon v.2.5.1 with Mascot server v.2.5 (Matrix Science). Search parameters and protein identifications are detailed in Supplementary Table S1.