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Glycosylation of proteins plays an important role in biological systems. The attachment of polysaccharide chains serves various key cellular functions. Thus, it is often critical to determine the exact site of glycosylation. Tandem mass spectrometry (MS/MS) employing linear ion trap technology has emerged as a cornerstone for protein identification and PTM analysis in bottom-up proteomics. However, the glycosylation moiety is usually fragile and falls off easily during the collision-induced dissociation (CID) MS/ MS activation process without leaving much evidence of its sites of attachment. Electron transfer dissociation (ETD) has recently been commercially introduced onto a linear ion trap. This new fragmentation methodology shows great promise in its ability to preserve the saccharide side chains on a peptide, while obtaining fragmentation between the α-carbon and the nitrogen throughout the backbone. This enables the exact glycosylation site to be deduced from the MS/MS spectrum.
N-linked glycopeptides from bovine fetuin and O-linked glycopeptides from a glycoprotein isolated from a Gram-positive bacterium were analyzed on a Finnigan LTQ with the added ETD functionality. While the O-linked glycans have residue masses around 400 Da, the N-linked glycan is well over 2800 Da. ETD MS/MS spectra showed contiguous c and z peptide fragment ion series preserving the sugar intact for N-linked peptides as well as O-linked peptides. The sites of the O-linked glycosylation deduced from the ETD MS/MS spectra have been confirmed with chemical degradation (beta-elimination) studies.