APPLICATION OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY AND MATRIX-ASSISTED LASER-DESORPTION IONIZATION TIME-OF-FLIGHT MASS-SPECTROMETRY IN COMBINATION WITH SELECTIVE ENZYMATIC MODIFICATIONS IN THE CHARACTERIZATION OF GLYCOSYLATION PATTERNS IN SINGLE-CHAIN PLASMINOGEN-ACTIVATOR

The application of high-performance liquid chromatography (HPLC), elec trospray ionization mass spectrometry (ESI-MS) and matrix-assisted las er-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-M S) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus sali vary plasminogen activator, a heterogeneous glycoprotein. The sample w as initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carb ohydrates or a combination of neuraminidase and O-glycosidase to remov e sialic acid and O-linked carbohydrates. By comparison of the LC-ESI- MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glyco sylation as well a number of N-linked glycopeptides. The O-linked glyc opeptides, which are present at a low level (<1%), were not detected p rior to the deglycosylation, nor could changes in peptide elution in t he map following deglycosylation be correlated with potential O-linked glycosylation sites.