Skip to main content
SpringerLink
Log in
Book cover

Glycosylation Engineering of Biopharmaceuticals pp 243–268Cite as

Noncovalent Mass Spectrometry for the Characterization of Antibody/Antigen Complexes

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 988))

Abstract

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need therefore to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their posttranslational modifications with the aim of reducing their chemistry, manufacturing, and control liabilities.

This chapter will provide the reader with a description of the general approach allowing antibody/antigen systems to be characterized by noncovalent MS. In the present chapter, we describe how recent noncovalent MS technologies are used to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) over-expressed in tumor cells. We will detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of noncovalent antibody/antigen complexes by MS. We will then focus on the type and the reliability of the information that we get from noncovalent MS data, with emphasis on the determination of the stoichiometry of antibody/antigen systems. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Beck A, Wurch T, Bailly C, Corvaia N (2010) Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol 10:345–352

    Article  PubMed  CAS  Google Scholar 

  2. Hager-Braun C, Katinger H, Tomer KB (2006) The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen. J Immunol 176:7471–7481

    PubMed  CAS  Google Scholar 

  3. Kiselar JG, Downard KM (1999) Direct identification of protein epitopes by mass spectrometry without immobilization of antibody and isolation of antibody-peptide complexes. Anal Chem 71:1792–1801

    Article  PubMed  CAS  Google Scholar 

  4. Pimenova T, Nazabal A, Roschitzki B, Seebacher J, Rinner O, Zenobi R (2008) Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry. J Mass Spectrom 43:185–195

    Article  PubMed  CAS  Google Scholar 

  5. Heck AJ (2008) Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods 5:927–933

    Article  PubMed  CAS  Google Scholar 

  6. Heck AJ, van den Heuvel RH (2004) Investigation of intact protein complexes by mass spectrometry. Mass Spectrom Rev 23:368–389

    Article  PubMed  CAS  Google Scholar 

  7. Loo JA (1997) Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom Rev 16:1–23

    Article  PubMed  CAS  Google Scholar 

  8. Loo JA (2000) Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes. Int J Mass Spectrom 200:175–186

    Article  CAS  Google Scholar 

  9. Bich C, Scott M, Panagiotidis A, Wenzel RJ, Nazabal A, Zenobi R (2008) Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem 375:35–45

    Article  PubMed  CAS  Google Scholar 

  10. Nazabal A, Wenzel RJ, Zenobi R (2006) Immunoassays with direct mass spectrometric detection. Anal Chem 78:3562–3570

    Article  PubMed  CAS  Google Scholar 

  11. Atmanene C, Wagner-Rousset E, Malissard M, Chol B, Robert A, Corvaia N et al (2009) Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. Anal Chem 81:6364–6373

    Article  PubMed  CAS  Google Scholar 

  12. Lu X, DeFelippis MR, Huang L (2009) Linear epitope mapping by native mass spectrometry. Anal Biochem 395:100–107

    Article  PubMed  CAS  Google Scholar 

  13. Oda M, Uchiyama S, Noda M, Nishi Y, Koga M, Mayanagi K et al (2009) Effects of antibody affinity and antigen valence on molecular forms of immune complexes. Mol Immunol 47:357–364

    Article  PubMed  CAS  Google Scholar 

  14. Oda M, Uchiyama S, Robinson CV, Fukui K, Kobayashi Y, Azuma T (2006) Regional and segmental flexibility of antibodies in interaction with antigens of different size. FEBS J 273:1476–1487

    Article  PubMed  CAS  Google Scholar 

  15. Tito MA, Miller J, Walker N, Griffin KF, Williamson ED, Despeyroux-Hill D et al (2001) Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach. Biophys J 81:3503–3509

    Article  PubMed  CAS  Google Scholar 

  16. Zhang Z, Pan H, Chen X (2009) Mass spectrometry for structural characterization of therapeutic antibodies. Mass Spectrom Rev 28:147–176

    Article  PubMed  CAS  Google Scholar 

  17. Beck A, Bussat MC, Zorn N, Robillard V, Klinguer-Hamour C, Chenu S et al (2005) Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells. J Chromatogr B Analyt Technol Biomed Life Sci 819:203–218

    Article  PubMed  CAS  Google Scholar 

  18. Vis H, Dobson CM, Robinson CV (1999) Selective association of protein molecules followed by mass spectrometry. Protein Sci 8:1368–1370

    Article  PubMed  CAS  Google Scholar 

  19. Rehder DS, Chelius D, McAuley A, Dillon TM, Xiao G, Crouse-Zeineddini J et al (2008) Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity. Biochemistry 47:2518–2530

    Article  PubMed  CAS  Google Scholar 

  20. Bobst CE, Abzalimov RR, Houde D, Kloczewiak M, Mhatre R, Berkowitz SA et al (2008) Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches. Anal Chem 80:7473–7481

    Article  PubMed  CAS  Google Scholar 

  21. Beck A, Klinguer-Hamour C, Bussat MC, Champion T, Haeuw JF, Goetsch L et al (2007) Peptides as tools and drugs for immunotherapies. J Pept Sci 13:588–602

    Article  PubMed  CAS  Google Scholar 

  22. Haeuw JF, Beck A (2004) Proteomics for development of immunotherapies. In: Hondermack H (ed) Proteomics: biomedical and pharmaceutical applications. Lille/Kluwer Academic, France/The Netherlands, pp 243–278

    Chapter  Google Scholar 

  23. Beck A, Wagner-Rousset E, Bussat MC, Lokteff M, Klinguer-Hamour C, Haeuw JF et al (2008) Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 9:482–501

    Article  PubMed  CAS  Google Scholar 

  24. Chowdhury SK, Katta V, Chait BT (1990) Probing conformational changes in proteins by mass spectrometry. J Am Chem Soc 112:9012–9013

    Article  CAS  Google Scholar 

  25. Qian J, Liu T, Yang L, Daus A, Crowley R, Zhou Q (2007) Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion. Anal Biochem 364:8–18

    Article  PubMed  CAS  Google Scholar 

  26. Bachmann MF, Kalinke U, Althage A, Freer G, Burkhart C, Roost H et al (1997) The role of antibody concentration and avidity in antiviral protection. Science 276:2024–2027

    Article  PubMed  CAS  Google Scholar 

  27. Usinger WR, Lucas AH (1999) Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides. Infect Immun 67:2366–2370

    PubMed  CAS  Google Scholar 

  28. Hensley P (1996) Defining the structure and stability of macromolecular assemblies in solution: the re-emergence of analytical ultracentrifugation as a practical tool. Structure 4:367–373

    Article  PubMed  CAS  Google Scholar 

  29. Neri D, Montigiani S, Kirkham PM (1996) Biophysical methods for the determination of antibody-antigen affinities. Trends Biotechnol 14:465–470

    Article  PubMed  CAS  Google Scholar 

  30. Griffey RH, Sannes-Lowery KA, Drader JJ, Ve M, Swayze EE, Hofstadler SA (2000) Characterization of low-affinity complexes between RNA and small molecules using electrospray ionization mass spectrometry. J Am Chem Soc 122:9933–9938

    Article  CAS  Google Scholar 

  31. Sannes-Lowery KA, Griffey RH, Hofstadler SA (2000) Measuring dissociation constants of RNA and aminoglycoside antibiotics by electrospray ionization mass spectrometry. Anal Biochem 280:264–271

    Article  PubMed  CAS  Google Scholar 

  32. Ayed A, Krutchinsky AN, Ens W, Standing KG, Duckworth HW (1998) Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 12:339–344

    Article  PubMed  CAS  Google Scholar 

  33. Gabelica V, Galic N, Rosu F, Houssier C, De Pauw E (2003) Influence of response factors on determining equilibrium association constants of non-covalent complexes by electrospray ionization mass spectrometry. J Mass Spectrom 38:491–501

    Article  PubMed  CAS  Google Scholar 

  34. Peschke M, Verkerk UH, Kebarle P (2004) Features of the ESI mechanism that affect the observation of multiply charged noncovalent protein complexes and the determination of the association constant by the titration method. J Am Soc Mass Spectrom 15:1424–1434

    Article  PubMed  CAS  Google Scholar 

  35. Babu KR, Moradian A, Douglas DJ (2001) The methanol-induced conformational transitions of beta-lactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 12:317–328

    Article  PubMed  CAS  Google Scholar 

  36. Dobo A, Kaltashov IA (2001) Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS. Anal Chem 73:4763–4773

    Article  PubMed  CAS  Google Scholar 

  37. Grandori R, Matecko I, Mayr P, Müller N (2001) Probing protein stabilization by glycerol using electrospray mass spectrometry. J Mass Spectrom 36:918–922

    Article  PubMed  CAS  Google Scholar 

  38. Konermann L, Douglas DJ (1997) Acid-induced unfolding of cytochrome c at different methanol concentrations: electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure. Biochemistry 36:12296–12302

    Article  PubMed  CAS  Google Scholar 

  39. Konermann L, Douglas DJ (1998) Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes. J Am Soc Mass Spectrom 9:1248–1254

    Article  PubMed  CAS  Google Scholar 

  40. Konermann L, Douglas DJ (1998) Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions. Rapid Commun Mass Spectrom 12:435–442

    Article  PubMed  CAS  Google Scholar 

  41. Sogbein OO, Simmons DA, Konermann L (2000) Effects of pH on the kinetic reaction mechanism of myoglobin unfolding studied by time-resolved electrospray ionization mass spectrometry. J Am Soc Mass Spectrom 11:312–319

    Article  PubMed  CAS  Google Scholar 

  42. Konermann L, Silva EA, Sogbein OF (2001) Electrochemically induced pH changes resulting in protein unfolding in the ion source of an electrospray mass spectrometer. Anal Chem 73:4836–4844

    Article  PubMed  CAS  Google Scholar 

  43. Liang Y, Du F, Sanglier S, Zhou BR, Xia Y, Van Dorsselaer A et al (2003) Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry, isothermal titration calorimetry, and fluorescence spectroscopy. J Biol Chem 278:30098–30105

    Article  PubMed  CAS  Google Scholar 

  44. Heam MT, Quirino JP, Whisstock J, Terabe S (2002) Thermal unfolding of proteins studied by coupled reversed-phase HPLC-electrospray ionization mass spectrometry techniques based on isotope exchange effects. Anal Chem 74:1467–1475

    Article  PubMed  Google Scholar 

  45. Loo JA, Edmonds CG, Udseth HR, Smith RD (1990) Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra. Anal Chem 62:693–698

    Article  PubMed  CAS  Google Scholar 

  46. Kostrewa D, Brockhaus M, D’Arcy A, Dale GE, Nelboeck P, Schmid G et al (2001) X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif. EMBO J 20:4391–4398

    Article  PubMed  CAS  Google Scholar 

  47. Prota AE, Campbell JA, Schelling P, Forrest JC, Watson MJ, Peters TR et al (2003) Crystal structure of human junctional adhesion molecule 1: implications for reovirus binding. Proc Natl Acad Sci U S A 100:5366–5371

    Article  PubMed  CAS  Google Scholar 

  48. Sanglier S, Atmanene C, Chevreux G, Dorsselaer AV (2008) Nondenaturing mass spectrometry to study noncovalent protein/protein and protein/ligand complexes: technical aspects and application to the determination of binding stoichiometries. Methods Mol Biol 484:217–243

    Article  PubMed  CAS  Google Scholar 

  49. Sanglier S, Ramstrom H, Haiech J, Leize E, Van Dorsselaer A (2002) Electrospray ionization mass spectrometry analysis revealed a ∼310 kDa noncovalent hexamer of HPr kinase/phosphatase from Bacillus subtilis. Int J Mass Spectrom 219:681–696

    Article  CAS  Google Scholar 

  50. Chernushevich IV, Thomson BA (2004) Collisional cooling of large ions in electrospray mass spectrometry. Anal Chem 76:1754–1760

    Article  PubMed  CAS  Google Scholar 

  51. Krutchinsky AN, Chernushevich IV, Spicer VL, Ens W, Standing KG (1998) Collisional damping interface for an electrospray ionization time-of-flight mass spectrometer. J Am Soc Mass Spectrom 9:569–579

    Article  CAS  Google Scholar 

  52. Schmidt A, Bahr U, Karas M (2001) Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS. Anal Chem 73:6040–6046

    Article  PubMed  CAS  Google Scholar 

  53. Sobott F, Hernandez H, McCammon MG, Tito MA, Robinson CV (2002) A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal Chem 74:1402–1407

    Article  PubMed  CAS  Google Scholar 

  54. Tahallah N, Pinkse M, Maier CS, Heck AJ (2001) The effect of the source pressure on the abundance of ions of noncovalent protein assemblies in an electrospray ionization orthogonal time-of-flight instrument. Rapid Commun Mass Spectrom 15:596–601

    Article  PubMed  CAS  Google Scholar 

  55. Kim MS, Lee SH, Song MY, Yoo TH, Lee BK, Kim YS (2007) Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms. J Mol Biol 374:1374–1388

    Article  PubMed  CAS  Google Scholar 

  56. Santora LC, Kaymakcalan Z, Sakorafas P, Krull IS, Grant K (2001) Characterization of noncovalent complexes of recombinant human monoclonal antibody and antigen using cation exchange, size exclusion chromatography, and BIAcore. Anal Biochem 299:119–129

    Article  PubMed  CAS  Google Scholar 

  57. Fuh G, Wu P, Liang WC, Ultsch M, Lee CV, Moffat B et al (2006) Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the AVASTIN(TM) Fab. J Biol Chem 281:6625–6631

    Article  PubMed  CAS  Google Scholar 

  58. Hernandez H, Robinson CV (2001) Dynamic protein complexes: insights from mass spectrometry. J Biol Chem 276:46685–46688

    Article  PubMed  CAS  Google Scholar 

  59. Robinson CV, Chung EW, Kragelund BB, Knudsen J, Aplin RT, Poulsen FM et al (1996) Probing the nature of noncovalent interactions by mass spectrometry. A study of protein–CoA ligand binding and assembly. J Am Chem Soc 118:8646–8653

    Article  CAS  Google Scholar 

  60. Smith RD, Light-Wahl KJ (1993) The observation of non-covalent interactions in solution by electrospray ionization mass spectrometry: Promise, pitfalls and prognosis. Biol Mass Spectrom 22:493–501

    Article  CAS  Google Scholar 

  61. Li YT, Hsieh YL, Henion JD, Senko MW, McLafferty FW, Ganem B (1993) Mass spectrometric studies on noncovalent dimers of leucine zipper peptides. J Am Chem Soc 115:8409–8413

    Article  CAS  Google Scholar 

  62. Li YT, Hsieh YL, Henion JD, Ocain TD, Schiehser GA, Ganem B (1994) Analysis of the energetics of gas-phase immunophilin-ligand complexes by ion spray mass spectrometry. J Am Chem Soc 116:7487–7493

    Article  CAS  Google Scholar 

  63. Darmanin C, Chevreux G, Potier N, Van Dorsselaer A, Hazemann I, Podjarny A et al (2004) Probing the ultra-high resolution structure of aldose reductase with molecular modelling and noncovalent mass spectrometry. Bioorg Med Chem 12:3797–3806

    Article  PubMed  CAS  Google Scholar 

  64. El Kabbani O, Rogniaux H, Barth P, Chung RP, Fletcher EV, Van Dorsselaer A et al (2000) Aldose and aldehyde reductases: correlation of molecular modeling and mass spectrometric studies on the binding of inhibitors to the active site. Proteins 41:407–414

    Article  PubMed  CAS  Google Scholar 

  65. Rogniaux H, Van Dorsselaer A, Barth P, Biellmann JF, Barbanton J, van Zandt M et al (1999) Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies. J Am Soc Mass Spectrom 10:635–647

    Article  PubMed  CAS  Google Scholar 

  66. Lofgren JA, Dhandapani S, Pennucci JJ, Abbott CM, Mytych DT, kaliyaperumal A et al (2007) Comparing ELISA and surface plasmon resonance for assessing clinical immunogenicity of panitumumab. J Immunol 178:7467–7472

    PubMed  CAS  Google Scholar 

  67. Orosz F, Ovadi J (2002) A simple method for the determination of dissociation constants by displacement ELISA. J Immunol Methods 270:155–162

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the CNRS, the University of Strasbourg UdS, and the Region Alsace. C.A. was supported by a studentship from the French Ministère de la Recherche.

Author information

Authors and Affiliations

  1. Laboratoire de Spectrométrie de Masse Bio-Organique, IPHC, CNRS, UMR7178, Université de Strasbourg, Strasbourg, France

    Cédric Atmanene, Alain Van Dorsselaer & Sarah Sanglier-Cianférani

  2. Physico-Chemistry Department, Centre d’Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France

    Elsa Wagner-Rousset

  3. Centre d’Immunologie Pierre-Fabre, Saint-Julien-en-Genevois, France

    Nathalie Corvaïa

  4. Antibody Physico-Chemistry Department, Centre d’Immunologie Pierre-Fabre, Saint Julien-en-Genevois, France

    Alain Beck

Authors
  1. Cédric Atmanene
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elsa Wagner-Rousset
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathalie Corvaïa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alain Van Dorsselaer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alain Beck
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sarah Sanglier-Cianférani
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. , Antibody Physico-Chemistry, Centre d'Immunologie Pierre Fabre (CIPF), Avenue Napoléon III 5, St. Julien-en-Genevois, 74164, France

    Alain Beck

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Atmanene, C., Wagner-Rousset, E., Corvaïa, N., Van Dorsselaer, A., Beck, A., Sanglier-Cianférani, S. (2013). Noncovalent Mass Spectrometry for the Characterization of Antibody/Antigen Complexes. In: Beck, A. (eds) Glycosylation Engineering of Biopharmaceuticals. Methods in Molecular Biology, vol 988. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-327-5_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-327-5_16

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-326-8

  • Online ISBN: 978-1-62703-327-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation