Anion supercharging enables structural assignment of oxidatively released O-glycans

O-glycan analysis faces analytical challenges due to poor fragmentation characteristics of oxidatively released O-glycan acids, which can preserve labile O-acetyl modifications but suffer from charge fixation at the reducing end. This study develops a supercharging approach using hexafluoroisopropanol/butylamine (HFIP/butylamine) mobile phase additives for enhanced mass spectrometric analysis of bleach-released O-glycan acids.
HFIP/butylamine increased average charge states by up to 50% compared to traditional mobile phases, dramatically improving MS2 fragmentation quality and enabling discrimination between isomeric compositions and structures. Enhanced fragmentation facilitated confident identification of sialic acid variants and acetylation patterns, overcoming the analytical bottleneck of poor acid fragmentation.

Computational optimization reduced database search times by up to 9-fold while maintaining sensitivity. Application to multi-species gastric mucins revealed distinct glycosylation profiles: porcine mucin showed predominant serine-linked neutral structures, while bovine and ovine mucins exhibited primarily threonine-linked sialylated glycans. A minor fraction of porcine and bovine mucins contained acetylated serine-linked sialylated glycans. This methodology provides a comprehensive framework for O-glycan characterization with preserved biological modifications.