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.