Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis
Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis
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DOI：
10.1016/s0021-9673(02)01357-2
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发表时间：
2002-12-20
影响因子：
4.1
通讯作者：
Prevelige, PE
中科院分区：
文献类型：
--
作者：
Lam, TT;Lanman, JK;Prevelige, PE
对于对于常规X射线衍射或核磁共振分析而言太大、不可结晶/不可溶或浓度低的蛋白质复合物，可以通过比较游离与复合的骨架酰胺氢的H/H-2交换速率（以及因此溶剂可及性）来绘制接触表面。蛋白质。首先将蛋白质暴露于（H2O）-H-2，允许在几个反应期的每一个中进行交换，然后用胃蛋白酶消化。H/H-2交换的程度和速率通过测量每种肽的质量随H/H-2交换时间的增加来确定。在这里，我们提出了一个实验方案，结合快速（最大限度地减少反交换）HPLC前端分离与超高分辨率质量分析（需要同时区分几十种肽的同位素分布）。该方法用于研究组装的人类免疫缺陷病毒型衣壳蛋白（CA）及其可溶性形式。(C)2002 Elsevier Science B. V.保留所有权利。
For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/H-2 exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed. protein(s). The protein is first exposed to (H2O)-H-2, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/H-2 exchange is determined by measuring the increase in mass with H/H-2 exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form. (C) 2002 Elsevier Science B.V. All rights reserved.