Fig. 2: Analysis of public SARS-CoV-2 Spike glycoproteome data.

a Site-specific N-glycan identification comparison using different software tools on a single N-glycopeptide dataset of the SARS-CoV-2 Spike protein. b Search speed comparison using the same N-glycopeptide data (PXD0018506³⁶). All searches were initiated from RAW files, using the same protein database. Notably, pGlycoNovo performed searches in a glycan search space 16–1000 times larger than the other four tools. Detailed comparison procedures are in Supplementary Note 2. c Expanded N-glycoproteome revealed by pGlycoNovo in public SARS-CoV-2 Spike Protein MS data (PXD0018506³⁶, 30 RAW files). Results were compared with those published using Byonic for the same data (Supplementary Note 3). d Distribution of site-specific N-glycans on the SARS-CoV-2 Spike protein. We performed statistical analysis of the 523 site-specific glycans identified by combining results from pGlycoNovo and the published data. Glycans were categorized into five groups based on monosaccharide composition: oligomannose type, hybrid type, complex type, truncated glycans, and unclassified type, where “unclassified type” refers to rare glycans not included in existing glycan database-dependent search engines (Supplementary Note 4). e Analysis of site-specific O-glycans using pGlycoNovo on SARS-CoV-2 Spike protein. It is noted that this public data does not include ETD spectra, making it impossible for us to differentiate between neighboring sites. f An annotated spectrum of an intact N-glycopeptide with a rare glycan attached. g An annotated spectrum of an intact O-glycopepide attached with a rare glycan attached. Peptide sequence with “J” indicating the N-glycosylation site. The glycan symbols are as follows: green circle for Hex, blue square for HexNAc, red triangle for fucose, yellow star for xylose, and color block diamond for HexA. Here, we used publicly available datasets (PXD0018506³⁶, totally 30 RAW files). In figure a, one RAW data file was used to compare the seven software tools (Supplementary Note 2). In figure b, six RAW data files were used to compare the five software tools, and additional search time comparisons were provided in Supplementary Note 2. In figures c and d, all 30 RAW files were used to identify an expanded glycoproteome dataset with rare glycans (Supplementary Note 3).