Anion Supercharging Enables Structural Analysis 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 introduces a supercharging approach using hexafluoroisopropyl alcohol (HFIP) and 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, improving the MS2 fragmentation quality 5-fold and enabling discrimination between isomeric compositions and structures. Enhanced fragmentation enabled confident identification of O -glycan sialic acid variants and acetylation patterns, overcoming the analytical bottleneck of poor O -glycan acid fragmentation. Computational optimization, representing modified monosaccharides as a single mass instead of individual masses, reduced database search times by up to 9-fold while maintaining sensitivity. Application to multispecies 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 while preserving biological modifications.