Enrichment and identification of glycopeptides in the hemostatic system. (A) Depiction of the proteomics workflow. Human platelet, plasma, and endothelial samples were reduced, alkylated, de-sialylated using neuraminidase, and subsequently digested using either chymotrypsin or trypsin. Glycopeptides were then enriched from the resulting complex peptide mixtures by sequential LWAC using VVA and PNA lectins. VVA enrichment was not used for plasma samples because of the absence of Tn glycans; however, plasma samples were further separated using isoelectric focusing to reduce sample complexity. Fractions containing glycopeptides were separated by online reverse-phase liquid chromatography followed by identification using Orbitrap FTMS. (B) Glycoproteins and glycosites identified in each sample in this study. (C) Overlap of the O-glycoproteins identified in this study with previously published O-glycoproteins. Left, O-glycoproteins identified in native samples. Right, overlap with all reported O-glycoproteins. (D) Sensitivity of LWAC detection. The abundance of each platelet O-glycoprotein was determined based on the platelet protein copy numbers reported in Burkhart et al³⁶  and plotted as a histogram (left) alongside the total platelet protein abundance obtained from the same study (right). Proteins with <500 copies are below the limit of quantification and are indicated by an asterisk. Comparison of the 2 distributions indicates that O-glycoproteins are detected over the full dynamic range of protein expression and include a similar proportion of proteins that are present below the limit of quantification. Note that membrane proteins are not quantified and therefore excluded from analysis. (E) Count of Ser-, Thr-, and Tyr-linked O-glycans. (F) Number of unambiguous glycosites per protein.