Derivatization of Sialylated Glycopeptides (DOSG) Enabling Site-Specific Isomeric Profiling Using LC-MS/MS

Jieqiang Zhong, Yifan Huang, Yehia Mechref

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


Glycosylation is a ubiquitous and important post-translational modification, functioning as a critical role in many biological processes. Among all the monosaccharides, sialic acids are of utmost importance, due to their relatively high abundance and association with various diseases. Elucidation of linkage isomers on sialic acids remains challenging. Various chemical derivatization techniques have been developed to identify the linkage isomers of sialic acids on released N-glycans, such as esterification and alkylamidation. Here, we present an efficient, effective, and stable derivatization method that can discriminate α2,3- and α2,6-linked sialic acids on glycopeptides by generating mass difference of the isomers. A similar chemical method has been developed for released N-glycans. We adapted and optimized the method to be effectively utilized for the derivatization of glycopeptides. The method also efficiently derivatized the carboxylic groups on peptides in a controlled way, which was confirmed by MS/MS data. The efficient derivatization of the peptide backbone carboxylic groups prompts ca. 4.6-fold increase in signal. The method was employed to characterize sialylated glycopeptides derived from bovine fetuin, human alpha-1 acid glycoprotein (AGP), and human erythropoietin (EPO). The method effectively and reliably allowed the characterization of sialylated N- and O-glycopeptide isomers. For example, 11 sialylated glycans including 28 isoforms were well profiled on Asn72 of AGP. The method is facile and highly reproducible that targets site- and sialic acid linkage-specific profiling of glycosylation.

Original languageEnglish
Pages (from-to)5763-5772
Number of pages10
JournalAnalytical Chemistry
Issue number14
StatePublished - Apr 13 2021


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