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

Nature Chemistry volume 6, pages 65–74 (2014)Cite this article

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An Addendum to this article was published on 21 March 2014

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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.

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Article 23 June 2022

Ieva Bagdonaite, Stacy A. Malaker, … Nichollas E. Scott

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  • 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.

References

  1. Hart, G. W. & Copeland, R. J. Glycomics hits the big time. Cell 143, 672–676 (2010).

    Article  CAS  Google Scholar 

  2. Moremen, K. W., Tiemeyer, M. & Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nature Rev. Mol. Cell Biol. 13, 448–462 (2012).

    Article  CAS  Google Scholar 

  3. Hanisch, F-G. Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionisation mass spectrometry for glycosylation site analysis. Anal. Chem. 83, 4829–4837 (2011).

    Article  CAS  Google Scholar 

  4. Kolarich, D., Lepenies, B. & Seeberger, P. H. Glycomics, glycoproteomics and the immune system. Curr. Opin. Chem. Biol. 16, 214–220 (2012).

    Article  CAS  Google Scholar 

  5. Antonopoulos, A., North, S. J., Haslam, S. M. & Dell, A. Glycosylation of mouse and human immune cells: insights emerging from N-glycome analyses. Biochem. Soc. Trans. 39, 1334–1340 (2011).

    Article  CAS  Google Scholar 

  6. Devakumar, A., Mechref, Y., Kang, P., Novotny, M. V. & Reilly J. P. Identification of isomeric N-glycan structures by mass spectrometry with 157 nm laser-induced photofragmentation. J. Am. Soc. Mass Spectrosc. 19, 1027–1040 (2008).

    Article  CAS  Google Scholar 

  7. Adibekian, A. et al. Comparative bioinformatics analysis of the mammalian and bacterial glycomes. Chem. Sci. 2, 337–344 (2011).

    Article  CAS  Google Scholar 

  8. Rini, J. M., Esko, J. D. & Varki, A. in Essentials of Glycobiology 2nd edn (eds Varki, A. et al.) Ch. 5 (Cold Spring Harbor Laboratory Press, 2009).

    Google Scholar 

  9. Gerken, T. A. et al. Emerging paradigms for the initiation of mucin-type protein O-glycosylation by the polypeptide GalNAc transferase family of glycosyltransferases. J. Biol. Chem. 286, 14493–14507 (2011).

    Article  CAS  Google Scholar 

  10. Laurent, N. et al. SPOT synthesis of peptide arrays on self-assembled monolayers and their evaluation as enzyme substrates. ChemBioChem. 9, 2592–2596 (2008).

    Article  CAS  Google Scholar 

  11. Laurent, N. et al. Enzymatic glycosylation of peptide arrays on gold surfaces. ChemBioChem. 9, 883–887 (2008).

    Article  CAS  Google Scholar 

  12. Su, J. & Mrksich, M. Using mass spectrometry to characterize self-assembled monolayers presenting peptides, proteins, and carbohydrates. Angew. Chem. Int. Ed. 41, 4715–4718 (2002).

    Article  CAS  Google Scholar 

  13. Errey, J. C., Mukhopadhyay, B., Kartha, R. & Field, R. A. Flexible enzymatic and chemo-enzymatic approaches to a broad range of uridine-diphospho-sugars. Chem. Commun. 2796–2707 (2004).

  14. Rejzek, M. et al. Chemical synthesis of UDP-Glc-2,3-diNAcA, a key intermediate in cell surface polysaccharide biosynthesis in the human respiratory pathogens B. pertussis and P. aeruginosa. Org. Biomol. Chem. 7, 1203–1210 (2009).

    Article  CAS  Google Scholar 

  15. Wandall, H. H. et al. Substrate specificities of three members of the human UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3. J. Biol. Chem. 272, 23503–23514 (1997).

    Article  CAS  Google Scholar 

  16. Ramakrishnan, B., Boeggeman, E. & Qasba P. K. Novel method for in vitro O-glycosylation of proteins: application for bioconjugation. Bioconjug. Chem. 18, 1912–1918 (2007).

    Article  CAS  Google Scholar 

  17. Jung, E., Gooley, A. A., Packer N. H., Karuso P. & Williams, K. L. Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum—in vivo studies on glycosylation of mucin Muc 1 and Muc 2 repeats. Eur. J. Biochem. 253, 517–524 (1998).

    Article  CAS  Google Scholar 

  18. Previato, J. O. et al. Biosynthesis of O-N-acetylglucosamine-linked glycans in Trypanosoma cruzi. J. Biol. Chem. 273, 14982–14988 (1998).

    Article  CAS  Google Scholar 

  19. Thoden, J. B., Wohlers, T. M., Fridovich-Keil, J. L. & Holden, H. M. Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site. J. Biol. Chem. 276, 15131–15136 (2001).

    Article  CAS  Google Scholar 

  20. Jansson, P. E., Kenne, L. & Widmalm, G. Computer-assisted structural analysis of polysaccharides with an extended version of CASPER using 1H- and 13C-N.M.R. data. Carbohydr. Res. 188, 169–191 (1989).

    Article  CAS  Google Scholar 

  21. Lundborg, M. & Widmalm, G. Structural analysis of glycans by NMR chemical shift prediction. Anal. Chem. 83, 1514–1517 (2011).

    Article  CAS  Google Scholar 

  22. Šardzík, R. et al. Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase. J. Am. Chem. Soc. 134, 4521–4524 (2012).

    Article  Google Scholar 

  23. Gagneux, P. & Varki, A. Evolutionary considerations in relating oligosaccharide diversity to biological function. Glycobiology 9, 747–755 (1999).

    Article  CAS  Google Scholar 

  24. Ercan, A. & West, C. M. Kinetic analysis of a golgi UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-α-glucosaminyltransferase from Dictyostelium. Glycobiology 15, 489–500 (2005).

    Article  CAS  Google Scholar 

  25. Taniguchi, N., Honke, K. & Fukuda, M. (eds) in Handbook of Glycosyltransferases and Related Genes Chs 9–14 (Springer-Verlag, 2003).

    Google Scholar 

  26. Lau, K. S. et al. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation. Cell 129, 123–134 (2007).

    Article  CAS  Google Scholar 

  27. Creese, A. J. & Cooper, H. J. Separation and identification of isomeric glycopeptides by high field asymmetric waveform ion mobility spectrometry. Anal. Chem. 84, 2597–2601 (2012).

    Article  CAS  Google Scholar 

  28. Li, H. et al. Resolving structural isomers of monosaccharide methyl glycosides using drift tube and travelling wave ion mobility mass spectrometry. Anal. Chem. 84, 3231–3239 (2012).

    Article  CAS  Google Scholar 

  29. Li, H., Bendiak, B., Siems, W. F., Gang, D. R. & Hill, H. H. Carbohydrate structure characterization by tandem ion mass spectroscopy (IMMS)2. Anal. Chem. 85, 2760–2769 (2013).

    Article  CAS  Google Scholar 

  30. Pagel, K. & Harvey, D. J. Ion mobility mass spectrometry of complex carbohydrates—collision cross sections of sodiated N-linked glycans. Anal. Chem. 85, 5138–5145 (2013).

    Article  CAS  Google Scholar 

  31. Slovin, S. F., Keding, S. J. & Ragupathi, G. Carbohydrate vaccines as immunotherapy for cancer. Immunol. Cell Biol. 83, 418–428 (2005).

    Article  CAS  Google Scholar 

  32. Kumar, S. R., Sauter, E. R., Quinn, T. P. & Deutscher, S. L. Thomsen-Friedenreich and Tn antigens in nipple fluid: carbohydrate biomarkers for breast cancer detection. Clin. Cancer Res. 11, 6868–6871 (2005).

    Article  CAS  Google Scholar 

  33. Hakomori, S. The glycosynapse. Proc. Natl Acad. Sci. USA 99, 225–232 (2002).

    Article  CAS  Google Scholar 

  34. Zaia, J. Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 23, 161–227 (2004).

    Article  CAS  Google Scholar 

  35. Borsdorf, H. & Eiceman, G. A. Ion mobility spectrometry: principles and applications. Appl. Spectrosc. Rev. 41, 323–375 (2006).

    Article  CAS  Google Scholar 

  36. Chawner, R. et al. QconCAT standard for calibration of ion mobility-mass spectrometry systems. J. Proteome Res. 11, 5564–5572 (2012).

    Article  CAS  Google Scholar 

  37. Wyttenbach, T., Bushnell, J. E. & Bowers, M. T. Salt bridge structures in the absence of solvent? The case for the oligoglycines. J. Am. Chem. Soc. 120, 5098–5103 (1998).

    Article  CAS  Google Scholar 

  38. Hurtado-Guerrero, R., Dorfmueller, H. C. & van Aalten, D. M. Molecular mechanisms of O-GlcNAcylation. Curr. Opin. Struct. Biol. 18, 551–557 (2008).

    Article  CAS  Google Scholar 

  39. Muller, R., Jenny, A. & Stanley, P. The EGF repeat-specific O-GlcNAc-transferase Eogt interacts with Notch signaling and pyrimidine metabolism pathways in Drosophila. PLoS ONE 8, e62835 (2013).

    Article  CAS  Google Scholar 

  40. Domon, B. & Costello, C. E. Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry. Biochemistry 27, 1534–1543 (1988).

    Article  CAS  Google Scholar 

Download references

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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Author notes

  1. S. L. Flitsch & C. E. Eyers

    Present address: Present addresses: Protein Function Group, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK (C.E.E.), School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK (S.L.F.),

  2. P. Both, A. P. Green and C. J. Gray: These authors contributed equally to this work

Authors and Affiliations

  1. School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

    P. Both, A. P. Green, C. J. Gray, R. Šardzík, M. Austeri, D. Richardson, S. L. Flitsch & C. E. Eyers

  2. College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095, China

    J. Voglmeir

  3. Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, S-106 91, Sweden

    C. Fontana & G. Widmalm

  4. Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK

    M. Rejzek & R. A. Field

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Contributions

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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