LC-MS/MS ANALYSIS OF SITE-OCCUPANCY AND SITE-SPECIFIC GLYCOSYLATION OF MULTIPLE-SITE GLYCOSYLATED PROTEINS

摘要

Complementary LC-MS and sample preparation technologies were integrated for systematic analyses of glycosylation and site occupancy. The methods were not only effective, but also efficient by searching identified glycans as optional modifications using the commercial protein characterization software Biophar-maLynx. These developments provide an approach for detailed characterization of glycoprotein therapeutics and vaccines (i.e. influenza and HIV) having complex glycosylation, which is often challenging. In addition, we also demonstrated: 1) Sensitive analysis of procainimide labeled glycans, including highly sialiced ones, on HILIC and mix-mode columns with different elution patterns; 2) RP-LC co-eluted glycopeptides could be further separated by HILIC-LC (more development on the way).