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Determination of Epitopes by Mass Spectrometry

  • Christine Hager-Braun
  • Kenneth B. Tomer
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 94)

Abstract

As a response to an infection, the immune system produces antibodies. The determination of the antigenic structure recognized by the antibody through epitope mapping provides information about the interaction between antigen and antibody for the diagnosis of a disease on a molecular level, for characterizing the pathogenesis of the infectious material, and for the development of interfering drugs or preventative vaccines. Here we present the determination of the fine structure of the linear epitope located on the gp41 protein of the human immunodeficiency virus recognized by the monoclonal antibody 2F5. In this approach we coupled the antigen SOSgp140 to the antibody 2F5, which was covalently linked to an Fc-specific antibody immobilized on cyanogen bromide (CNBr)-activated Sepharose beads. Digestion of the antigen with endoproteinase LysC resulted in an affinity-bound peptide whose fine structure was characterized by digestion with carboxypeptidase Y and aminopeptidase M. All steps of this method were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). The epitope recognized by 2F5 was identified to be the 16-mer peptide with the sequence NEQELLELDKWASLWN.

Keywords

Human Immunodeficiency Virus Nuclear Magnetic Resonance Spectroscopy Cyanogen Bromide Slow Rotation Sepharose Bead 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    World Health Organization’s AIDS Epidemic Update Report for December 2002 as found on the internet: http://www.unaids.org/barcelona/presskit/barcelona%20report/contents_html.html.
  2. 2.
    Parker, C. E., Deterding, L. J., Hager-Braun, C., et al. (2001) Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5. J. Virol. 75, 10,906–10,911.PubMedCrossRefGoogle Scholar
  3. 3.
    Peter, J. F. and Tomer, K. B. (2001) A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and crosslinking. Anal. Chem. 73, 4012–4019.PubMedCrossRefGoogle Scholar
  4. 4.
    Zwick, M., Labrijn, A. F., Wang, M., et al. (2001) Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J. Virol. 75, 10,892–10,905.PubMedCrossRefGoogle Scholar
  5. 5.
    Reineke, U., Ivascu, C., Schlief, M., et al. (2002) Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences. J. Immunol. Methods 267, 37–51.PubMedCrossRefGoogle Scholar
  6. 6.
    Wegner, G. J., Lee, H. J., and Corn, R. M. (2002) Characterization and optimization of peptide arrays for the study of epitope-antibody interaction using surface plasmon resonance imaging. Anal. Chem. 74, 5161–5168.PubMedCrossRefGoogle Scholar
  7. 7.
    Thali, M., Moore, J. P., Furman, C., et al. (1993) Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J. Virol. 67, 3978–3988.PubMedGoogle Scholar
  8. 8.
    Trkola, A., Purtschner, M., Muster, T., et al. (1996) Human monoclonal antibody 2G12 defines a distinctive neutralizing epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70, 1100–1108.PubMedGoogle Scholar
  9. 9.
    Jemmerson, R. and Paterson, Y. (1986) Mapping epitopes on a protein antigen by the proteolysis of antigen-antibody complexes. Science 232, 1001–1004.PubMedCrossRefGoogle Scholar
  10. 10.
    Jensen, T. H., Jensen, A., Szilvay, A. M., and Kjems, J. (1997) Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites. FEBS Lett. 414, 50–54.PubMedCrossRefGoogle Scholar
  11. 11.
    Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W. A. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659.PubMedCrossRefGoogle Scholar
  12. 12.
    Sharon, M., Gorlach, M., Levy, R., Hayek, Y., and Anglister, J. (2002) Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies. Protein Expres. Purif. 24, 374–383.CrossRefGoogle Scholar
  13. 13.
    Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Epitope mapping of the gastrin releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption mass spectrometry. Protein Sci. 3, 1488–1492.CrossRefGoogle Scholar
  14. 14.
    Parker, C. E., Papac, D. I., Trojak, S. K., and Tomer, K. B. (1996) Epitope mapping by mass spectrometry: determination of an epitope on HIV-1IIIB p26 recognized by a monoclonal antibody. J. Immunol. 15, 198–206.Google Scholar
  15. 15.
    Jeyarajah, S., Parker, C. E., Sumner, M. T., and Tomer, K. B. (1998) MALDI/MS mapping of HIV-gp120 epitopes recognized by a limited polyclonal antibody. J. Am. Soc. Mass Spectrom. 9, 157–165.PubMedCrossRefGoogle Scholar
  16. 16.
    Schülke, N., Vesanen, M. S., Sanders, R. W., et al. (2002) Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein. J. Virol. 76, 7760–7776.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Christine Hager-Braun
    • 1
  • Kenneth B. Tomer
    • 1
  1. 1.Department of Health and Human ServicesLaboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of HealthResearch Triangle Park

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