Glycosylation inhibits proteolytic processing. In vitro cleavage analysis of glycopeptides and their nonglycosylated counterparts. Synthetic peptides were in vitro–glycosylated and then subjected to a protease digestion time course with the indicated enzymes. (A) Summary of results. Identified cleavage sites are indicated by an arrow. Proteases affected by glycosylation are colored blue (partial inhibition) or red (complete inhibition). Glycosites identified by prior glycoproteomic analysis are shown in red; glycosites identified by LC-MS/MS on in vitro glycosylated peptides are indicated by a yellow box. Glycosylation of the TFPI1_2 peptide both delayed and repositioned NE cleavage. (B) Example matrix-assisted laser desorption/ionization–time-of-flight spectra showing glycoform-dependent inhibition of thrombin serine protease activity. The monoisotopic mass of the cleavage product is indicated in red. NE, neutrophil elastase; THRB, thrombin; PLMN, Plasmin. n = 2.