Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents

Protein O-glycosylation is one of themost diversepost-translational modifications. A critical step in the analysisof O-glycomes is the release of glycans from glycoconjugates.Current release methods rely mainly on beta-elimination, whichcan result in peeling reactions and loss of base-sensitive functionalitiesleading to misidentification of glycans. To address this challenge,well-defined synthetic glycopeptides were used to establish a robustworkflow for the oxidative release of O-glycans suitablefor glycomics. Treatment of O-glycopeptides withneutralized hypochlorite resulted in the selective formation of lactic/glycolicacid glycosides, thereby retaining unique information of the parentamino acid (serine/threonine) that is lost by beta-elimination.It locks the glycan in a closed ring configuration, thereby preventingpeeling, and furthermore, the carboxylate of the anomeric tag promotesionization in negative ion mode mass spectrometry, thereby increasingsignal intensities. Labile modifications such as sialic acids, sulfates,and acetyl esters are maintained during the release procedure. Thepromise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identifiedmono- and di-O-acetylated sialoglycans as well aspreviously undetected tri-O-acetylated and sulfatedglycans. The use of well-defined glycopeptide standards made it alsopossible to identify reaction intermediates, which in turn allowedus to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.