Summary
The ability to accurately characterize an epitope on an antigen is essential to understand the pathogenesis of an infectious material, and for the design and development of drugs and vaccines. Emergence of a new contagious microbial or viral variant necessitates the need for robust identification and characterization of the antigenic determinant. Recent advances have made mass spectrometry (MS) a robust and sensitive analytical tool with high mass accuracy. The use of MS to characterize peptides and proteins has gained popularity in the research arena involving protein–protein interactions. Combining the modern mass spectrometric principles of protein–protein interaction studies with the classical use of limited proteolysis, a linear epitope on a peptide or a protein antigen can be accurately mapped in a short time, compared with other traditional techniques available for epitope mapping. Additionally, complete MS analyses can be achieved with very little sample consumption. Here we discuss the overall approach to characterize the detailed interaction between a linear antigen (either a peptide or a protein antigen) and its corresponding monoclonal antibody by using MS. The steps involved in epitope excision, epitope extraction, and indirect immunosorption are outlined thoroughly. Conditions required for MS analysis using either matrix assisted laser desorption ionization (MALDI) or electrospray ionization (ESI) sources are summarized, with special emphasis on the experimental protocols.
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References
Kuby, J. (ed.) (1997) Immunology. W.H. Freeman and Co., New York, NY.
Hager-Braun, C., and Tomer, K. B. (2005) Determination of protein-derived epitopes by mass spectrometry. Expert Rev. Proteomics 2, 745–756.
Laver, W. G., Air, G. M., Webster, R. G., and Smithgill, S. J. (1990) Epitopes on protein antigens: misconceptions and realities. Cell 61, 553–556.
Harlow, E., and Lane, D. (eds.) (1988) Anti-bodies–a laboratory manual. Cold Spring Harbor, New York, NY, pp. 23–36.
Zhu, C. S., Liu, X. S., Feng, J. N., Zhang, W., Shen, B. F., Ou’yang, W., Cao, Y. X., and Jin, B. Q. (2006) Characterization of the neutralizing activity of three anti-human TNF monoclonal antibodies and prediction of their TNF epitopes by molecular modeling and mutant protein approach. Immunol. Lett. 102, 177–183.
Cerutti, M. L., Ferreiro, D. U., Sanguineti, S., Goldbaum, F. A., and de Prat-Gay, G. (2006) Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2. Biochemistry 45, 15520–15528.
Bentley, G. A., Bhat, T. N., Boulot, G., Fischmann, T., Navaza, J., Poljak, R. J., Riottot, M. M., and Tello, D. (1989) Immunochemical and crystallographic studies of antibody D1.3 in its free, antigen-liganded, and idiotope-bound states. Cold Spring Harb. Sym. 54, 239–245.
Lescar, J., Stouracova, R., Riottot, M. M., Chitarra, V., Brynda, J., Fabry, M., Horejsi, M., Sedlacek, J., and Bentley, G. A. (1997) Three-dimensional structure of an Fab–peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody. J. Mol. Biol. 267, 1207–1222.
Padlan, E. A. (1994) Anatomy of the antibody molecule. Mol. Immunol. 31, 169–217.
Padlan, E. A., Abergel, C., and Tipper, J. P. (1995) Identification of specificity-determining residues in antibodies. FASEB J. 9, 133–139.
Tormo, J., Blaas, D., Parry, N. R., Rowlands, D., Stuart, D., and Fita, I. (1994) Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2. EMBO J. 13, 2247–2256.
Hager-Braun, C., Katinger, H., and Tomer, K. B. (2006) The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen. J. Immunol. 176, 7471–7481.
Papac, D. I., Hoyes, J., and Tomer, K. B. (1994) Epitope mapping of the gastrin-releasing peptide/antibombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. Protein Sci. 3, 1485–1492.
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-1 IIIB p26 recognized by a monoclonal antibody. J. Immunol. 157, 198–206.
Parker, C. E., and Tomer, K. B. (2002) MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies. Mol. Biotechnol. 20, 49–62.
Peter, J. F., and Tomer, K. B. (2001) A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking. Anal. Chem. 73, 4012–4019.
Purcell, A. W., and Gorman, J. J. (2001) The use of post-source decay in matrix-assisted laser desorption/ionisation mass spectrometry to delineate T cell determinants. J. Immunol. Methods 249, 17–31.
Purcell, A. W., and Gorman, J. J. (2004) Immunoproteomics: mass spectrometry-based methods to study the targets of the immune response. Mol. Cell Proteomics 3, 193–208.
Purcell, A. W., Zeng, W. G., Mifsud, N. A., Ely, L. K., MacDonald, W. A., and Jackson, D. C. (2003) Dissecting the role of peptides in the immune response: theory, practice and the application to vaccine design. J. Pept. Sci. 9, 255–281.
Anglister, J., Scherf, T., Zilber, B., Levy, R., Zvi, A., Hiller, R., and Feigelson, D. (1993) Two-dimensional NMR investigations of the interactions of antibodies with peptide antigens. FASEB J. 7, 1154–1162.
Scherf, T., and Anglister, J. (1993) A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens. Biophys. J. 64, 754–761.
Butler, J. E. (2000) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4–23.
Mullett, W. M., Lai, E. P. C., and Yeung, J. M. (2000) Surface plasmon resonance-based immunoassays. Methods 22, 77–91.
Wang, L. F., and Yu, M. (2004) Epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics. Curr. Drug Targets 5, 1–15.
Williams, J. G., Tomer, K. B., Hioe, C. E., Zolla-Pazner, S., and Norris, P. J. (2006) The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 1560–1569.
Jemmerson, R., and Blankenfeld, R. (1989) Affinity consideration in the design of synthetic vaccines intended to elicit antibodies. Mol. Immunol. 26, 301–307.
Parham, P. (1983) On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice. J. Immunol. 131, 2895–2902.
Borchers, C., and Tomer, K. B. (1999) Characterization of the noncovalent complex of human immunodeficiency virus glycoprotein 120 with its cellular receptor CD4 by matrix-assisted laser desorption/ionization mass spectrometry. Biochemistry 38, 11734–11740.
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64–71.
Loo, J. A. (2000) Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes. Int. J. Mass Spectrom. 200, 175–186.
Sobott, F., and Robinson, C. V. (2002) Proteincomplexes gain momentum. Curr. Opin. Struct. Biol. 12, 729–734.
Tanaka, K. (2003) The origin of macromole-cule ionization by laser irradiation. Angew. Chem. Int. Edit. 42, 3860–3870.
Suckau, D., Kohl, J., Karwath, G., Schneider, K., Casaretto, M., Bittersuermann, D., and Przybylski, M. (1990) Molecular epitope identification by limited proteolysis of an immobilized antigen–antibody complex and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. USA 87, 9848–9852.
Macht, M., Marquardt, A., Deininger, S. O., Damoc, E., Kohlmann, M., and Przybylski, M. (2004) “Affinity-proteomics”: direct protein identification from biological material using mass spectrometric epitope mapping. Anal. Bioanal. Chem. 378, 1102–1111.
Suckau, D., Mak, M., and Przybylski, M. (1992) Protein surface topology-probing by selective chemical modification and mass spectrometric peptide mapping. Proc. Natl. Acad. Sci. USA 89, 5630–5634.
Acknowledgments
We thank Dr. Leesa J. Deterding for helpful discussions. This work was supported by Intramural Research program, National Institute of Environmental Health Sciences, NIH.
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Dhungana, S., Williams, J.G., Fessler, M.B., Tomer, K.B. (2009). Epitope Mapping by Proteolysis of Antigen–Antibody Complexes. In: Schutkowski, M., Reineke, U. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 524. Humana Press. https://doi.org/10.1007/978-1-59745-450-6_7
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DOI: https://doi.org/10.1007/978-1-59745-450-6_7
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