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.