Characterizing the glycosylation of pectin degrading enzymes by mass spectrometry
Colangelo, Jennifer Lynn White
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The pectin degrading enzymes produced by the fungus Aspergillus niger have many commercial applications and are important for the study of plant pathogenesis. Current research on these enzymes has focused on their structure to gain an understanding of their biological activities. The glycosylation of these enzymes is of interest because it is known to affect the biochemical properties of proteins, as well as their biological activity. Therefore, characterizing the glycosylation of pectin degrading enzymes may provide insight into their biological roles. Mass spectrometric analyses in conjunction with biochemical techniques were implemented to characterize the glycosylation of various recombinant pectin degrading enzymes. For example, the endoproteinase digestion of a glycoprotein can be analyzed by liquid chromatography/mass spectrometry in conjunction with in-source collision induced dissociation to identify glycopeptides in the digestion mixture. Once identified, the glycopeptides can be analyzed by matrix assisted laser desorption/ionization (MALDI)-mass spectrometry with on-target glycosidase digestions, a recently developed technique for determining the primary sequence of glycans. The glycosylation of endopolygalacturonase I, endopolygalacturonase II, pectate lyase, and pectin methylesterase was characterized. The N-linked sites were found occupied with high mannose glycans, and the results indicated that pectin methylesterase contained some hybrid structures. No evidence of O-linked glycosylation was observed. During their characterization, steps were taken to refine the procedure for on-target glycosidase digestions to save both time and materials. The time required for digestion was reduced from hours to minutes, and the digestions were performed at room temperature instead of elevated temperatures. Some endoglycosidases were found to retain their activity in the presence of the MALDI matrix, sinapinic acid. By performing the digestion in the matrix, only one aliquot of sample was needed for analysis.