Epitope mapping by differential chemical modification of antigens.
Matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry combined with differential chemical modification have proven to be versatile tools for epitope mapping as well as for studying diverse protein-protein and protein-ligand interactions. Characterization of a discontinuous or a conformational epitope on an antigen demands the ability to map the three-dimensional protein surface along with the interface of two interacting proteins. Classical methods of differentially derivatizing amino acid residues have been successfully merged with highly sensitive and highly accurate mass spectrometric techniques to rapidly profile the three-dimensional protein surface and determine the surface accessibility of specific amino acid residues. Here we discuss the use of mass spectrometry to characterize discontinuous or conformational epitopes by studying antigen-antibody interactions. The steps involved in epitope mapping approaches using differential chemical modification and H/D exchange on the antigen are discussed in detail, with particular emphasis on the experimental protocols.
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, 111 T.W. Alexander Drive, PO Box 12233, Research Triangle Park, NC 27709, USA.
Suraj
S
Dhungana
Michael B
MB
Fessler
Kenneth B
KB
Tomer
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D010447:Peptide Hydrolases