Epitope Mapping by Proteolysis of Antigen–Antibody Complexes

  • Suraj Dhungana
  • Jason G. Williams
  • Michael B. Fessler
  • Kenneth B. Tomer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 524)


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 words

Antibody 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.


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Suraj Dhungana
    • 1
  • Jason G. Williams
    • 1
  • Michael B. Fessler
    • 1
  • Kenneth B. Tomer
    • 1
  1. 1.Laboratory of Structural BiologyNational Institute of Environmental Health Sciences, NIH, DHHSResearch Triangle ParkUSA

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