Demonstration of hydrazide tagging for O-glycans and a central composite design of experiments optimization using the INLIGHT? reagent

摘要

AbstractThe INLIGHT? strategy forN-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT? strategy and adopted it for use withO-linked glycans. A central composite design was utilized to find optimized tagging conditions (45 acetic acid, 0.1?μg/μL tag concentration, 37 C, 1.75?h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging ofO-linked glycans for the first time.Graphical abstractThe INLIGHT? hydrazide tag was optimized using a central composite design for derivatization ofO-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT? derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.>