Epitope mapping by amide hydrogen/deuterium exchange coupled with immobilization of antibody, on-line proteolysis, liquid chromatography and mass spectrometry

被引：87

作者：

Coales, Stephen J. ^{[1
]}

Tuske, Steven J. ^{[1
]}

Tornasso, Justine C. ^{[1
]}

Hamuro, Yoshitomo ^{[1
]}

机构：

[1] ExSAR Corp, Monmouth Jct, NJ 08852 USA

来源：

RAPID COMMUNICATIONS IN MASS SPECTROMETRY | 2009年 / 23卷 / 05期

关键词：

HYDROGEN-EXCHANGE; PROTEIN-STRUCTURE; BINDING-SITES; ANTIGEN; DYNAMICS; COMPLEX; SUBUNIT; LIGAND;

D O I：

10.1002/rcm.3921

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 (C) 2009 John Wiley & Sons, Ltd.

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页码：639 / 647

页数：9

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