ProteinO-glycosylation is one of the
most diverse
post-translational modifications. A critical step in the analysis
ofO-glycomes is the release of glycans from glycoconjugates.
Current release methods rely mainly on β-elimination, which
can result in peeling reactions and loss of base-sensitive functionalities
leading to misidentification of glycans. To address this challenge,
well-defined synthetic glycopeptides were used to establish a robust
workflow for the oxidative release ofO-glycans suitable
for glycomics. Treatment ofO-glycopeptides with
neutralized hypochlorite resulted in the selective formation of lactic/glycolic
acid glycosides, thereby retaining unique information of the parent
amino acid (serine/threonine) that is lost by β-elimination.
It locks the glycan in a closed ring configuration, thereby preventing
peeling, and furthermore, the carboxylate of the anomeric tag promotes
ionization in negative ion mode mass spectrometry, thereby increasing
signal intensities. Labile modifications such as sialic acids, sulfates,
and acetyl esters are maintained during the release procedure. The
promise of the approach was demonstrated by the analysis ofO-glycans from bovine submaxillary mucin, which identified
mono- and di-O-acetylated sialoglycans as well as
previously undetected tri-O-acetylated and sulfated
glycans. The use of well-defined glycopeptide standards made it also
possible to identify reaction intermediates, which in turn allowed
us to postulate a reaction mechanism for oxidativeO-glycan release under neutral conditions.