Fig. 3: N-Glycoproteome profiling with pGlycoNovo and pGlyco3 across five evolutionarily distant species.
a Overall workflow of intact glycopeptide profiling in five species. b Number of identified glycopeptide spectra in each species. c, Number of identified glycoproteins and site-specific glycans in each species. d Contribution of glycopeptide spectra identified by pGlycoNovo and pGlyco3. e Contribution of site-specific glycans identified by pGlycoNovo and pGlyco3. f Classification of site-specific glycans in each species by glycan type. Glycans were categorized into five groups based on their 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). g Distribution of specific monosaccharide-containing glycopeptides in each species. h An annotated spectrum of a glycopeptide with four fucoses. The peptide sequence with “J” indicating the N-glycosylation site. The glycan symbols are as follows: green circle for Hex, blue square for HexNAc and red triangle for fucose. i The workflows of the comprehensive 13C/15N isotopic-labeling strategy for the FDR validation. j Validation results from the isotopically labeled fission yeast and A. thaliana. The element-level error rate (incorrect number of N or C elements) of the identified glycopeptides was tested via the 15N-/13C-labeled precursor signals. Data are presented as mean values ± SD. Each bar represents the average value across biological replicates. Yeast experiments include three biological replicates, and plant experiments include two biological replicates. Each point represents an individual measurement, with error bars indicating the SD.
