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通过酰胺氢/氘交换、抗体固定、在线蛋白酶解、液相色谱和质谱联用进行表位作图。

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

作者信息

Coales Stephen J, Tuske Steven J, Tomasso Justine C, Hamuro Yoshitomo

机构信息

ExSAR Corporation, 11 Deer Park Drive, Suite 103, Monmouth Junction, NJ 08852, USA.

出版信息

Rapid Commun Mass Spectrom. 2009 Mar;23(5):639-47. doi: 10.1002/rcm.3921.

Abstract

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.

摘要

利用酰胺氢/氘(H/D)交换结合固定化抗体、在线胃蛋白酶水解、液相色谱(LC)和质谱(MS)技术,确定了马细胞色素c针对单克隆抗体E8的表位。结果与通过E8-细胞色素c复合物的X射线共晶体结构鉴定的接触残基以及通过核磁共振(NMR)光谱进行H/D交换获得的结果总体上高度一致。细胞色素c的H/D交换反应在有或没有固定化E8抗体的情况下进行。通过H/D交换质谱法,在有抗体存在时比没有抗体时获得较少氘的区域被定义为表位。精心设计了对照实验,以帮助高可信度地鉴定表位。

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