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

Journal name:
Nature Chemistry
Volume:
6,
Pages:
65–74
Year published:
DOI:
doi:10.1038/nchem.1817
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. Examples of common epimeric glycoconjugates and families of enzymes involved in their biosynthesis.
    Figure 1: Examples of common epimeric glycoconjugates and families of enzymes involved in their biosynthesis.

    a, The pp-α-GanT family of enzymes mediate the transfer of an α-linked GalNAc residue to the hydroxyl groups of serine and threonine residues in proteins. This modification is common in higher eukaryotes (including humans) and represents the first step in the biosynthesis of mucin-type O-glycans. b, In unicellular eukaryotes, mucin-type O-glycans have been found initiated with an α-linked GlcNAc residue generated by a pp-α-GnT family of enzymes. c, β-linked GlcNAc is a common post-translational modification (PTM) observed in many organisms and is especially prevalent among multicellular eukaryotes. The attachment of this residue is mediated by O-GlcNAc transferase (OGT)38. β-linked GlcNAc is also transferred to the notch epidermal growth factor repeats by extracellular OGT (ref. 39). The differentiation of epimeric glycopeptides by mass spectrometry has not been reported previously.

  2. Application of peptide microarrays to investigate the sugar donor promiscuity of pp-α-GanT2.
    Figure 2: Application of peptide microarrays to investigate the sugar donor promiscuity of pp-α-GanT2.

    a, Peptides are immobilized on a SAM on a gold platform through the N-terminus of the peptide by formation of an amide bond. The immobilized peptides are then incubated with pp-α-GanT2 and the relevant NDP-sugar donor, and the reaction is monitored by MALDI-ToF MS. b, Table of peptide sequences 114 immobilized with their parent protein (in parentheses). The activated sugar donors screened are listed along the top of the table (for full structures see Supplementary Fig. 1). Positive activity, as detected by MS, is denoted with +.

  3. Synthesis and characterization of epimeric glycopeptides 15 and 16.
    Figure 3: Synthesis and characterization of epimeric glycopeptides 15 and 16.

    a, Treatment of peptide 14 with ppGanT2 and either UDP-GlcNAc or UDP-GalNAc results in the synthesis of glycopeptides 15 and 16, respectively. bg, Selected regions from the 1H,1H-TOCSY spectra (τmix = 100 ms) of 15 (b) and 16 (c) showing the spin systems originating from the anomeric protons, and selected regions from the 1H,1H-NOESY spectra (τmix = 300 ms) of 15 (d,e) and 16 (f,g) showing, inter alia, inter-residue correlations from the anomeric protons to H3 and H4 in the corresponding threonine residues.

  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 4: TWIMS ATDs showing the discrimination of epimeric glycopeptides 15–17 and 19,20 and the distinction of HexNAc oxonium ions generated following CID.

    a,b, Overlaid ATDs showing that glycopeptide 15 (α-GlcNAc, red) can be distinguished from glycopeptide 16 (α-GalNAc, blue) (a) and that glycopeptide 16 (α-GalNAc, blue) can be distinguished from glycopeptide 17 (β-GlcNAc, black) (b). c, CID of glycopeptides 15, 16 and 17 (α-GalNAc/α-GlcNAc/β-GlcNAc) before IM separation results in neutral loss of the glycan moiety, leaving the unmodified peptide, which cannot be differentiated by IM-MS (as would be expected). d, Overlaid ATDs associated with both the glycosylated Muc 2 (PTTTPITTTTTVTPTPTPTGTQT) species 19 and 20, demonstrating the separation of multiple glycoforms, which can be partially resolved under the conditions used. The more abundant conformations of the epimeric glycopeptides can still be differentiated by TWIMS-MS by differences in their drift times. e, CID of glycopeptides 19 and 20 before IM separation results in the formation of epimeric oxonium ions that are distinguishable by TWIMS-MS. Vertical dashed lines represent drift time as identified by ½ fwhh. fwhh, full width half height.

  5. Drift times of disaccharides 27–31 and the monosaccharide product ions generated following CID.
    Figure 5: Drift times of disaccharides 27–31 and the monosaccharide product ions generated following CID.

    a, CID of a disaccharide commonly results in the formation of B, C, Y and Z ions, which arise as a result of a single bond cleavage40. The combination of CID and TWIMS allows the drift times and CCS values of these product ions to be determined. b, Structures of the β-1,4-linked disaccharides 2731 and representative structures of the monosaccharide product ions (highlighted in red) formed as a result of CID of the respective disaccharide precursor ion. Drift times for the [M+Na]+ species and calculated CCS values are shown below each structure. For cases where two conformations (potential sites of sodiation) were observed, the drift time and CCS measurements are reported for the predominant species. CCS values were determined following calibration using polyglycine peptides with known CCS values37. CCS values for the disaccharides were not determined (ND), as drift time values lie outside this calibration range.

  6. Sequencing the Man3 glycan (33) and the related Man1 glycan (34) using IM-MS of product ions generated by ISD and/or 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.

    Drift times (in ms) and CCS values (in Å2) in parentheses for selected [M+Na]+ product ions are presented below the structures. Terminal monosaccharide and disaccharide product ions could be characterized after a single round of CID. Internal residues were characterized following ISD, quadrupole mass isolation of the desired ISD product ion, and subsequent CID before IM-MS. C1 ions can be grouped either as β-Man (shaded dark green) or α-Man (shaded light green). All identified Y1, C2 and Y2 product ions could be characterized as GlcNAc (shaded blue), Manβ1-4GlcNAc (shaded red) and GlcNAcβ1-4GlcNAc (shaded purple), respectively. For cases where two conformations (potential sites of sodiation) were observed, the drift time and CCS measurements are reported for the predominant species. CCS values were determined following calibration using polyglycine with known CCS values37. CCS values for the disaccharides were not determined (ND) as the drift time values lie outside this calibration range. n/a, not applicable.

Change history

Corrected online 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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Author information

  1. These authors contributed equally to this work

    • P. Both,
    • A. P. Green &
    • C. J. Gray

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, S-106 91 Stockholm, 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
  5. 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.)

    • S. L. Flitsch &
    • C. E. Eyers

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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