Epitope Mapping by Proteolysis of Antigen–Antibody Complexes
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.
Key wordsAntibody Antigen Epitope mapping Linear epitope Epitope excision and extraction Limited proteolysis Epitope foot-printing Mass spectrometry MALDI-MS ESI-MS
We thank Dr. Leesa J. Deterding for helpful discussions. This work was supported by Intramural Research program, National Institute of Environmental Health Sciences, NIH.
- 1.Kuby, J. (ed.) (1997) Immunology. W.H. Freeman and Co., New York, NY.Google Scholar
- 4.Harlow, E., and Lane, D. (eds.) (1988) Anti-bodies–a laboratory manual. Cold Spring Harbor, New York, NY, pp. 23–36.Google Scholar
- 5.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.PubMedCrossRefGoogle Scholar
- 8.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.PubMedCrossRefGoogle Scholar
- 25.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.PubMedCrossRefGoogle Scholar
- 33.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.PubMedCrossRefGoogle Scholar