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 CHARACTERIZA

摘要

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove 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 glycosylation as well a number of N-linked glycopeptides. The O-linked glycopeptides, which are present at a low level (<1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.