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

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Carbohydrate-Based Vaccines

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1331))

Abstract

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.

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Abbreviations

2-AA:

2-Aminobenzoic acid (anthranilic acid)

2-AB:

2-Aminobenzamide

BHK:

Baby hamster kidney

CCD:

Charge-coupled device

CID:

Collision-induced dissociation

DC-SIGN:

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

DHB:

2,5-Dihydroxybenzoic acid

DMSO:

Dimethylsulfoxide

DMT-MM:

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

EDTA:

Ethylenediaminetetraacetate

ESI:

Electrospray ionization

Fuc:

Fucose

Gal:

Galactose

GC/MS:

Gas chromatography/mass spectrometry

GlcNAc:

N-acetylglucosamine

GMEM:

Glasgow’s Minimum Essential Medium

H20N100E2:

20 mM HEPES 100 mM NaCl, 2 mM EDTA

HEPES:

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

HIV:

Human immunodeficiency virus

HPLC:

High-performance liquid chromatography

MALDI:

Matrix-assisted laser desorption/ionization

Man:

Mannose

MoI:

Multiplicity of infection

MS:

Mass spectrometry

Neu5Ac:

N-acetylneuraminic acid (sialic acid)

Neu5Gc:

N-glycoylneuraminic acid

PCR:

Polymerase chain reaction

PGC:

Porous graphitized carbon

PNGase F:

Protein-N-glycosidase F

PBS:

Phosphate-buffered saline

Q:

Quadrupole

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

THAP:

2,4,6-Trihydroxyacetophenone

TOF:

Time-of-flight

Tris-base:

2-Amino-2-(hydroxymethyl)-1,3-propanediol

UUKV:

Uukuniemi virus

References

  1. Bowden TA, Jones EY, Stuart DI (2011) Cells under siege: viral glycoprotein interactions at the cell surface. J Struct Biol 175:120–126

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Rossmann MG (2013) Structure of viruses: a short history. Q Rev Biophys 46:133–180

    Article  CAS  PubMed  Google Scholar 

  3. Chang VT, Crispin M, Aricescu AR et al (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15:267–273

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Lozach PY, Kuhbacher A, Meier R et al (2011) DC-SIGN as a receptor for phleboviruses. Cell Host Microbe 10:75–88

    Article  CAS  PubMed  Google Scholar 

  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 402:187–196

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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:e23521

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 107:13800–13805

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Walker LM, Huber M, Doores KJ et al (2011) Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477:466–470

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 76:7306–7321

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Kornfeld R, Kornfeld S (1985) Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 54:631–664

    Article  CAS  PubMed  Google Scholar 

  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 326:285–289

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 85:9998–10009

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 40:657–668

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Crispin M, Bowden TA (2013) Antibodies expose multiple weaknesses in the glycan shield of HIV. Nat Struct Mol Biol 20:771–772

    Article  CAS  PubMed  Google Scholar 

  15. Dalziel M, Crispin M, Scanlan CN et al (2014) Emerging principles for the therapeutic exploitation of glycosylation. Science 343:1235681

    Article  PubMed  Google Scholar 

  16. Burton DR, Ahmed R, Barouch DH et al (2012) A blueprint for HIV vaccine discovery. Cell Host Microbe 12:396–407

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 84:6208–6217

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 88:10244–10251

    Article  PubMed Central  PubMed  Google Scholar 

  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 10:1027–1032

    Article  CAS  PubMed  Google Scholar 

  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 23:303–312

    Article  CAS  PubMed  Google Scholar 

  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 82:8300–8306

    Article  CAS  PubMed  Google Scholar 

  22. Harvey DJ (2005) Fragmentation of negative ions from carbohydrates: Part 2, Fragmentation of high-mannose N-linked glycans. J Am Soc Mass Spectrom 16:631–646

    Article  CAS  PubMed  Google Scholar 

  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 16:622–630

    Article  CAS  PubMed  Google Scholar 

  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 16:647–659

    Article  CAS  PubMed  Google Scholar 

  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 376:44–60

    Article  CAS  PubMed  Google Scholar 

  26. Domon B, Costello CE (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj J 5:397–409

    Article  CAS  Google Scholar 

  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 23:1955–1966

    Article  CAS  PubMed  Google Scholar 

  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 34:2368–2378

    Article  CAS  PubMed  Google Scholar 

  29. Pettersson R, Kääriäinen L (1973) The ribonucleic acids of Uukuniemi virus, a noncubical tick-borne arbovirus. Virol J 56:608–619

    Article  CAS  Google Scholar 

  30. Lozach PY, Mancini R, Bitto D et al (2010) Entry of bunyaviruses into mammalian cells. Cell Host Microbe 7:488–499

    Article  CAS  PubMed  Google Scholar 

  31. Pettersson R, Kääriäinen L, von Bonsdorff CH et al (1971) Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 46:721–729

    Article  CAS  PubMed  Google Scholar 

  32. Grassucci RA, Taylor D, Frank J (2008) Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat Protoc 3:330–339

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Cyrklaff M, Roos N, Gross H et al (1994) Particle surface interaction in thin vitrified films for cryo-electron microscopy. J Microsc 175:135–142

    Article  CAS  Google Scholar 

  34. Crispin M, Harvey DJ, Bitto D et al (2014) Structural plasticity of the Semliki Forest virus glycome upon interspecies transmission. J Proteome Res 13:1702–1712

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  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 9:1031–1034

    Article  Google Scholar 

  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 26:469–479

    Article  CAS  PubMed  Google Scholar 

  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 22:1047–1052

    Article  CAS  PubMed  Google Scholar 

  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 72:5027–5039

    Article  CAS  PubMed  Google Scholar 

  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 55:13159–13170

    Article  CAS  Google Scholar 

  40. Morelle W, Michalski JC (2007) Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2:1585–1602

    Article  CAS  PubMed  Google Scholar 

  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 83:2492–2499

    Article  CAS  PubMed  Google Scholar 

  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 215–244

    Google Scholar 

  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 28:2008–2018

    Article  CAS  PubMed  Google Scholar 

  44. Cooper CA, Gasteiger E, Packer NH (2001) GlycoMod - a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 1:340–349

    Article  CAS  PubMed  Google Scholar 

  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 45:815–819

    Article  CAS  PubMed  Google Scholar 

  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 18:2357–2359

    Article  CAS  PubMed  Google Scholar 

  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 9:3796–3801

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

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

Author information

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK

    David Bitto

  2. Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK

    David J. Harvey & Laura K. Pritchard

  3. Division of Structural Biology, University of Oxford, Oxford, UK

    Steinar Halldorsson

  4. King’s College London, School of Medicine at Guy’s, King’s and St Thomas’ Hospitals, Guy’s Hospital, Great Maze Pond, London, UK

    Katie J. Doores

  5. Division of Structural Biology, University of Oxford, Oxford, UK

    Juha T. Huiskonen

  6. Division of Structural Biology, University of Oxford, Oxford, UK

    Thomas A. Bowden

  7. Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Max Crispin

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  1. David Bitto
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Correspondence to Max Crispin .

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Editors and Affiliations

  1. Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Hannover, Germany

    Bernd Lepenies

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Bitto, D. et al. (2015). Determination of N-linked Glycosylation in Viral Glycoproteins by Negative Ion Mass Spectrometry and Ion Mobility. In: Lepenies, B. (eds) Carbohydrate-Based Vaccines. Methods in Molecular Biology, vol 1331. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2874-3_7

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  • DOI: https://doi.org/10.1007/978-1-4939-2874-3_7

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2873-6

  • Online ISBN: 978-1-4939-2874-3

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