Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.
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This research was supported by grants from the Engineering and Physical Sciences Research Council (EPSRC), the Biotechnology and Biological Sciences Research Council (BBSRC), Swiss Scheme Foundation (MA), the Royal Society (Wolfson Award to S.L.F.), the Knut and Alice Wallenberg Foundation, the Swedish Research Council and the European commission (FP7). Work at John Innes Centre (JIC) was supported by a BBSRC Institute Strategic Programme Grant (BB/J004561/1) and the John Innes Foundation.
The authors declare no competing financial interests.
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Both, P., Green, A., Gray, C. et al. Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing. Nature Chem 6, 65–74 (2014) doi:10.1038/nchem.1817
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