Fig 1: Mapping the O-glycopeptidome reveals O-glycan-mediated cleavage (A) Unique O-glycopeptide IDs that are singly glycosylated (maroon) or multiply glycosylated (orange) across LACRT, PROL1, PROL4. (B) Protein-specific O-glycosylation landscape where Hexose, HexNAc, and Neu5Ac is represented by H, N, and A, respectively. Colors include maroon (N1), orange (N2), peach (H1N1A1), and dark blue (H2N2). (C) Distribution and occupancy of glycosites across major tear glycoproteins (LACRT n = 36 peptides; PROL1 n = 8; PROL4 n = 25). Kernel Density Estimation (KDE) of glycosite distance to the nearest terminus, weighted by AUC intensity (top, left). Peptide length and glycosite distance normalization formula (top, right) is shown. A value of 0.5 indicates a glycosite in the middle of a peptide sequence whereas a value of 0.0 indicates an N-/C-terminal glycosite. AUC-weighted KDE of normalized glycosite distances for both occupied and unoccupied sites separated by precursor protein (bottom). (D) Bar graph displaying relative % AUC intensities of unmodified and glycosylated (H1N1A1) PROL4 peptides where each point represents the value from one sample and the height of the bar represents the average across three. The relative AUC intensity (%) for all unmodified peptide backbones (gray) will add up to 100%; the same is true for glycopeptides (maroon). A two-tailed t test was used to evaluate statistical significance in the relative % intensities of a given backbone sequence with and without glycosylation. * denotes 0.01 < p < 0.05, ** denotes 0.001 < p < 0.01, and *** denotes 0.0001 < p < 0.001.