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We have evaluated O-glycosylation on the S1 and S2 subunits of SARS-CoV-2 spike protein by searching LC-MS/MS data for common O-glycosylation modifications. Interestingly, our O-glycoproteomic profiling indicated O-glycosylation at sites Thr323 and Ser325 on the S1 subunit of SARS-CoV-2 spike protein (Figures 3, 6 and 7). As O-glycosylation at Thr323 and Ser325 on the spike protein has not been reported before and is not indicated based on Cryo-EM data of SARS-CoV-2 S protein, we evaluated the detected O-glycopeptide manually (Walls et al. 2020). We observed very strong evidence for the presence of O-glycosylation at site Thr323 as b and y ions of the peptide 320VQPTESIVR328 with high mass accuracy were present. Upon manual validation of the fragment ions of O-glycopeptide 320VQPTESIVR328, we observed that Thr323 is the predominantly occupied site. This conclusion was based on the b (b1- m/z 228.13) and y (y1- m/z 175.12, y2- m/z 274.19, y4- 474.30, y5- 603.34 m/z and y7 + glycan- 1748.77 m/z) ions we detected upon fragmentation of the glycopeptide (Figure 7a). In addition, neutral losses and the detection of oxonium ions also confirmed the presence of glycosylation on these peptides. Core-1 mucin type O-glycans such as GalNAc, GalNAcGal and GalNAcGalNeuAc2 and Core-2 glycans GalNAcGalNeuAc(GlcNAcGal) and GalNAcGalNeuAc(GlcNAcGalNeuAc) were observed on site Thr323 (Figure 7). Possibly, Ser325 is occupied with HexNAcHexNeuAc glycan together with T323 (Figure 6), but this could not be confirmed unambiguously as electron transfer dissociation fragmentation on this peptide was not successful because of lower charge states of peptide (Supplementary Figures S3 and S4) (Shajahan et al. 2017).