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Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

An Addendum to this article was published on 21 March 2014

This article has been updated

Abstract

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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Figure 1: Examples of common epimeric glycoconjugates and families of enzymes involved in their biosynthesis.
Figure 2: Application of peptide microarrays to investigate the sugar donor promiscuity of pp-α-GanT2.
Figure 3: Synthesis and characterization of epimeric glycopeptides 15 and 16.
Figure 4: TWIMS ATDs showing the discrimination of epimeric glycopeptides 15–17 and 19,20 and the distinction of HexNAc oxonium ions generated following CID.
Figure 5: Drift times of disaccharides 27–31 and the monosaccharide product ions generated following CID.
Figure 6: Sequencing the Man3 glycan (33) and the related Man1 glycan (34) using IM-MS of product ions generated by ISD and/or CID.

Change history

  • 21 March 2014

    After this Article went to press the authors realized that a number of the key references had been inadvertently omitted or removed before the final submission of the manuscript. The authors would therefore like to cite the following additional articles:  1. Zhu, M. L., Bendiak, B., Clowers, B. & Hill, H. H. Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions. Anal. Bioanal. Chem. 394, 1853–1867 (2009). Structural characterization of select isomeric oligosaccharides using atmospheric ion-mobility spectrometry for separation of linkage and branch isomers, anomeric isomers, and epimers, prior to MS3 analysis using an ion-trap mass spectrometer. 2. Williams, J. P. et al. Characterization of simple isomeric oligosaccharides and the rapid separation of glycan mixtures by ion mobility mass spectrometry. Int. J. Mass Spectrom. 298, 119–127 (2010). Using both travelling-wave ion-mobility spectrometry and drift-tube ion-mobility spectrometry, released N-glycans and isobaric glycans were separated for subsequent characterization by tandem MS. Theoretical modelling was also used to confirm experimentally determined collisional cross-section values. 3. Fenn, L. S. & McLean, J. A. Structural resolution of carbohydrate positional and structural isomers based on gas-phase ion mobilitymass spectrometry. Phys. Chem. Chem. Phys. 13, 2196–2205 (2011). Details the collisional cross-section values of 300 sodiated positional and structural carbohydrate isomers from MALDI IM-MS. 4. Harvey, D. J. et al. Travelling wave ion mobility and negative ion fragmentation for the structural determination of N-linked glycans. Electrophoresis 34, 2368–2378 (2013).  Structural characterization of released N-glycans using negative-ion-mode collision-induced dissociation of ion-mobility-separated isomer (and conformer) precursors.

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Acknowledgements

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.

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C.E.E., S.L.F., A.P.G., P.B. and C.J.G. conceived the project, designed the experiments, discussed the results and implications, and commented on the manuscript at all stages. A.P.G., C.J.G., P.B., S.L.F. and C.E.E. co-wrote the paper. P.B., A.P.G. and C.J.G. contributed equally. C.J.G. performed the IM-MS experiments and statistical analysis. P.B., J.V. and D.R. performed the molecular biology and protein purification. P.B. and A.P.G. carried out kinetic studies and bioinformatics. A.P.G. performed the glycan chemical synthesis. R.Š., J.V. and M.A. carried out peptide synthesis and biotransformations in the solid phase and in solution. C.F. and G.W. performed and reported the NMR analysis. M.R. and R.A.F. synthesized the activated sugar donors. All authors commented on the manuscript.

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Correspondence to S. L. Flitsch or C. E. Eyers.

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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). https://doi.org/10.1038/nchem.1817

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