Determination of N-linked Glycosylation in Viral Glycoproteins by Negative Ion Mass Spectrometry and Ion Mobility

  • David Bitto
  • David J. Harvey
  • Steinar Halldorsson
  • Katie J. Doores
  • Laura K. Pritchard
  • Juha T. Huiskonen
  • Thomas A. Bowden
  • Max Crispin
Part of the Methods in Molecular Biology book series (MIMB, volume 1331)


Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans.

Key words

Virus Glycosylation Structure Mass spectrometry Glycoprotein 



2-Aminobenzoic acid (anthranilic acid)




Baby hamster kidney


Charge-coupled device


Collision-induced dissociation


Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin


2,5-Dihydroxybenzoic acid




4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride




Electrospray ionization






Gas chromatography/mass spectrometry




Glasgow’s Minimum Essential Medium


20 mM HEPES 100 mM NaCl, 2 mM EDTA


4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid


Human immunodeficiency virus


High-performance liquid chromatography


Matrix-assisted laser desorption/ionization




Multiplicity of infection


Mass spectrometry


N-acetylneuraminic acid (sialic acid)


N-glycoylneuraminic acid


Polymerase chain reaction


Porous graphitized carbon

PNGase F

Protein-N-glycosidase F


Phosphate-buffered saline




Sodium dodecylsulfate-polyacrylamide gel electrophoresis








Uukuniemi virus



M.C. is supported by the Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant (UM1AI100663) and the International AIDS Vaccine Initiative through the Neutralizing Antibody Consortium and Bill and Melinda Gates Center for Vaccine Discovery. M.C. is a Fellow of Oriel College, Oxford. We also thank the Wellcome Trust (grant number 090532/Z/09/Z), the Academy of Finland (grant numbers 130750 and 218080 to J.T.H.), and the MRC (MR/L009528/1 to T.A.B. and MR/K024426/1 to K.J.D) for funding. The Wellcome Trust Centre for Human Genetics is supported by Wellcome Trust Centre grant 090532/Z/09/Z


  1. 1.
    Bowden TA, Jones EY, Stuart DI (2011) Cells under siege: viral glycoprotein interactions at the cell surface. J Struct Biol 175:120–126PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Rossmann MG (2013) Structure of viruses: a short history. Q Rev Biophys 46:133–180PubMedCrossRefGoogle Scholar
  3. 3.
    Chang VT, Crispin M, Aricescu AR et al (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15:267–273PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Lozach PY, Kuhbacher A, Meier R et al (2011) DC-SIGN as a receptor for phleboviruses. Cell Host Microbe 10:75–88PubMedCrossRefGoogle Scholar
  5. 5.
    Alexandre KB, Gray ES, Lambson BE et al (2010) Mannose-rich glycosylation patterns on HIV-1 subtype C gp120 and sensitivity to the lectins, Griffithsin, Cyanovirin-N and Scytovirin. Virology 402:187–196PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bonomelli C, Doores KJ, Dunlop DC et al (2011) The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade. PLoS One 6:e23521PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Doores KJ, Bonomelli C, Harvey DJ et al (2010) Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc Natl Acad Sci U S A 107:13800–13805PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Walker LM, Huber M, Doores KJ et al (2011) Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477:466–470PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Scanlan CN, Pantophlet R, Wormald MR et al (2002) The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1 → 2 mannose residues on the outer face of gp120. J Virol 76:7306–7321PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Kornfeld R, Kornfeld S (1985) Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54:631–664PubMedCrossRefGoogle Scholar
  11. 11.
    Walker LM, Phogat SK, Chan-Hui PY et al (2009) Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326:285–289PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Bonsignori M, Hwang KK, Chen X et al (2011) Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors. J Virol 85:9998–10009PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Falkowska E, Le KM, Ramos A et al (2014) Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers. Immunity 40:657–668PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Crispin M, Bowden TA (2013) Antibodies expose multiple weaknesses in the glycan shield of HIV. Nat Struct Mol Biol 20:771–772PubMedCrossRefGoogle Scholar
  15. 15.
    Dalziel M, Crispin M, Scanlan CN et al (2014) Emerging principles for the therapeutic exploitation of glycosylation. Science 343:1235681PubMedCrossRefGoogle Scholar
  16. 16.
    Burton DR, Ahmed R, Barouch DH et al (2012) A blueprint for HIV vaccine discovery. Cell Host Microbe 12:396–407PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Bowden TA, Crispin M, Harvey DJ et al (2010) Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly. J Virol 84:6208–6217PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Crispin M, Harvey DJ, Bitto D et al (2014) Uukuniemi phlebovirus assembly and secretion leave a functional imprint on the virion glycome. J Virol 88:10244–10251PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Powell AK, Harvey DJ (1996) Stabilisation of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun Mass Spectrom 10:1027–1032PubMedCrossRefGoogle Scholar
  20. 20.
    Wheeler SF, Domann P, Harvey DJ (2009) Derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2-3) and α(2-6) isomers. Rapid Commun Mass Spectrom 23:303–312PubMedCrossRefGoogle Scholar
  21. 21.
    Liu X, Qiu H, Lee RK et al (2010) Methylamidation for sialoglycomics by MALDI-MS: a facile derivatization strategy for both α2,3- and α2,6-linked sialic acids. Anal Chem 82:8300–8306PubMedCrossRefGoogle Scholar
  22. 22.
    Harvey DJ (2005) Fragmentation of negative ions from carbohydrates: Part 2, Fragmentation of high-mannose N-linked glycans. J Am Soc Mass Spectrom 16:631–646PubMedCrossRefGoogle Scholar
  23. 23.
    Harvey DJ (2005) Fragmentation of negative ions from carbohydrates: Part 1; Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates. J Am Soc Mass Spectrom 16:622–630PubMedCrossRefGoogle Scholar
  24. 24.
    Harvey DJ (2005) Fragmentation of negative ions from carbohydrates: Part 3, Fragmentation of hybrid and complex N-linked glycans. J Am Soc Mass Spectrom 16:647–659PubMedCrossRefGoogle Scholar
  25. 25.
    Harvey DJ, Royle L, Radcliffe CM et al (2008) Structural and quantitative analysis of N-linked glycans by MALDI and negative ion nanospray mass spectrometry. Anal Biochem 376:44–60PubMedCrossRefGoogle Scholar
  26. 26.
    Domon B, Costello CE (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj J 5:397–409CrossRefGoogle Scholar
  27. 27.
    Harvey DJ, Scarff CA, Crispin M et al (2012) MALDI-MS/MS with traveling wave ion mobility for the structural analysis of N-linked glycans. J Am Soc Mass Spectrom 23:1955–1966PubMedCrossRefGoogle Scholar
  28. 28.
    Harvey DJ, Scarff CA, Edgeworth M et al (2013) Travelling wave ion mobility and negative ion fragmentation for the structural determination of N-linked glycans. Electrophoresis 34:2368–2378PubMedCrossRefGoogle Scholar
  29. 29.
    Pettersson R, Kääriäinen L (1973) The ribonucleic acids of Uukuniemi virus, a noncubical tick-borne arbovirus. Virol J 56:608–619CrossRefGoogle Scholar
  30. 30.
    Lozach PY, Mancini R, Bitto D et al (2010) Entry of bunyaviruses into mammalian cells. Cell Host Microbe 7:488–499PubMedCrossRefGoogle Scholar
  31. 31.
    Pettersson R, Kääriäinen L, von Bonsdorff CH et al (1971) Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 46:721–729PubMedCrossRefGoogle Scholar
  32. 32.
    Grassucci RA, Taylor D, Frank J (2008) Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat Protoc 3:330–339PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Cyrklaff M, Roos N, Gross H et al (1994) Particle surface interaction in thin vitrified films for cryo-electron microscopy. J Microsc 175:135–142CrossRefGoogle Scholar
  34. 34.
    Crispin M, Harvey DJ, Bitto D et al (2014) Structural plasticity of the Semliki Forest virus glycome upon interspecies transmission. J Proteome Res 13:1702–1712PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Börnsen KO, Mohr MD, Widmer HM (1995) Ion exchange and purification of carbohydrates on a Nafion(R) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun Mass Spectrom 9:1031–1034CrossRefGoogle Scholar
  36. 36.
    Domann P, Spencer DIR, Harvey DJ (2012) Production and fragmentation of negative ions from neutral N-linked carbohydrates ionized by matrix-assisted laser desorption/ionization. Rapid Commun Mass Spectrom 26:469–479PubMedCrossRefGoogle Scholar
  37. 37.
    Harvey DJ, Crispin M, Scanlan C et al (2008) Differentiation between isomeric triantennary N-linked glycans by negative ion tandem mass spectrometry and confirmation of glycans containing galactose attached to the bisecting (β1-4-GlcNAc) residue in N-glycans from IgG. Rapid Commun Mass Spectrom 22:1047–1052PubMedCrossRefGoogle Scholar
  38. 38.
    Wheeler SF, Harvey DJ (2000) Negative ion mass spectrometry of sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by matrix-assisted laser desorption/ionization-time-of-flight and electrospray-time-of-flight mass spectrometry. Anal Chem 72:5027–5039PubMedCrossRefGoogle Scholar
  39. 39.
    Kunishima M, Kawachi C, Morita J et al (1999) 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride: an efficient condensing agent leading to the formation of amides and esters. Tetrahedron 55:13159–13170CrossRefGoogle Scholar
  40. 40.
    Morelle W, Michalski JC (2007) Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2:1585–1602PubMedCrossRefGoogle Scholar
  41. 41.
    Selman MHJ, Hemayatkar M, Deelder AM et al (2011) Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides. Anal Chem 83:2492–2499PubMedCrossRefGoogle Scholar
  42. 42.
    Hossain M, Limbach PA (2010) A comparison of MALDI matrices. In: Cole RB (ed) Electrospray and MALDI mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications, 2nd edn. John Wiley and Sons Inc., Hoboken, NJ, pp 215–244Google Scholar
  43. 43.
    Harvey DJ, Edgeworth M, Krishna BA et al (2014) Fragmentation of negative ions from N-linked carbohydrates: Part 6: Glycans containing one N-acetylglucosamine in the core. Rapid Commun Mass Spectrom 28:2008–2018PubMedCrossRefGoogle Scholar
  44. 44.
    Cooper CA, Gasteiger E, Packer NH (2001) GlycoMod - a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 1:340–349PubMedCrossRefGoogle Scholar
  45. 45.
    Harvey DJ, Rudd PM (2010) Identification of by-products formed during the release of N-glycans with protein N-glycosidase F in the presence of dithiothreitol. J Mass Spectrom 45:815–819PubMedCrossRefGoogle Scholar
  46. 46.
    Omtvedt LA, Royle L, Husby G et al (2004) Artefacts formed by addition of urea to N-linked glycans released with peptide-N-glycosidase F for analysis by mass spectrometry. Rapid Commun Mass Spectrom 18:2357–2359PubMedCrossRefGoogle Scholar
  47. 47.
    Harvey DJ, Merry AH, Royle L et al (2009) Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 9:3796–3801PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • David Bitto
    • 1
  • David J. Harvey
    • 2
  • Steinar Halldorsson
    • 3
  • Katie J. Doores
    • 4
  • Laura K. Pritchard
    • 2
  • Juha T. Huiskonen
    • 5
  • Thomas A. Bowden
    • 6
  • Max Crispin
    • 7
  1. 1.Division of Structural Biology, Wellcome Trust Centre for Human GeneticsUniversity of OxfordOxfordUK
  2. 2.Oxford Glycobiology Institute, Department of BiochemistryUniversity of OxfordOxfordUK
  3. 3.Division of Structural BiologyUniversity of OxfordOxfordUK
  4. 4.King’s College London, School of Medicine at Guy’s, King’s and St Thomas’ Hospitals, Guy’s HospitalLondonUK
  5. 5.Division of Structural BiologyUniversity of OxfordOxfordUK
  6. 6.Division of Structural BiologyUniversity of OxfordOxfordUK
  7. 7.Oxford Glycobiology Institute, Department of BiochemistryUniversity of OxfordOxfordUK

Personalised recommendations