Fig. 6. O-glycopeptides are highly abundant in the ascites enrichment.
A Mucin-domain glycoproteins harbor many O-glycosites. O-glycopeptides identified by O-Pair Search are shown in red circles in approximate locations on the protein backbone; proteins were annotated based on Uniprot assignments (N-glycan sites, green) and the mucin-domain candidacy algorithm (mucin domain, red). B O-glycopeptide identifications map to mucin-domain glycoproteins identified with the mucin candidate algorithm. Protein identifications from O-Pair glycopeptide searches were analyzed with the mucin-domain candidacy algorithm, and this bar graph shows the percentage of protein identifications from the O-Pair Search that were non-mucin proteins (green) and mucin-domain glycoproteins (red). C O-glycopeptide PSMs are higher in elution samples. The total number of N- and O-glycopeptides were summed for each sample. The percentage of total glyco peptide spectral matches (PSMs) that were either N- or O-glycopeptides are shown in green and red, respectively. In both panels B and C, values represent the average of the 5 patient samples, error bars show one standard deviation, and percentages for each of the five samples are shown as data points on the bar graph. Glycopeptide-glycan networks in panels D and E compare N- and O-glycopeptides, respectively, in ascites and enriched samples. For each glycopeptide-glycan network, unique glycopeptides (i.e., peptide sequence and total glycan mass) are organized vertically in the middle, and unique total glycan masses are nodes arranged in the outer semi-circles. The left and right semi-circles are mirror images of each other, showing the same glycan masses in the same order on either side, as indicated by numbers. The left side (gray) shows which glycan masses map to glycopeptides from ascites samples, and the right side (green or red) shows which glycan masses map to glycopeptides from enriched samples. These figures indicate which glycopeptides are shared or unique between the unenriched and enriched conditions and show that O-glycopeptides are detected more often than N-glycopeptides in StcEE447D-enriched ascites fluid, the inverse of the non-enriched ascites fluid. Supplementary Data 11 and 12 provide total glycan mass compositions (outer nodes) identities and the unique glycopeptide list (middle nodes) for N-glycopeptide (D) and O-glycopeptide (E) networks, respectively. Source data are in Supplementary Data 10–13 and in Source Data.