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.