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Epitope mapping by amide hydrogen/deuterium exchange coupled with immobilization of antibody, on‐line proteolysis, liquid chromatography and mass spectrometry
Author(s) -
Coales Stephen J.,
Tuske Steven J.,
Tomasso Justine C.,
Hamuro Yoshitomo
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3921
Subject(s) - chemistry , hydrogen–deuterium exchange , mass spectrometry , chromatography , epitope , amide , monoclonal antibody , deuterium , epitope mapping , cytochrome c , antibody , biochemistry , peptide sequence , physics , quantum mechanics , gene , immunology , biology , mitochondrion
The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on‐line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X‐ray co‐crystal structure of the E8–cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence. Copyright © 2009 John Wiley & Sons, Ltd.