The INLIGHT™strategy for N-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
with O-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 of O-linked glycans for the first time.

EB00000002957K

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