Biochemistry of Macromolecular Interaction Unit, Department of Structural Biology and Chemistry, Institut Pasteur, CNRS UMR3528, Paris, France.
Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK.
Methods Mol Biol. 2020;2127:339-358. doi: 10.1007/978-1-0716-0373-4_22.
Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of "MS-friendly" detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from "MS-friendly" detergents.
整合膜蛋白参与多种生物功能，是重要的药物靶点。尽管它们在人类蛋白质组中大量存在，但整合膜蛋白结构在蛋白质数据库中的数量却大大不足。人们熟知此类生物系统的生物物理特性分析所面临的挑战。大多数结构方法，包括 X 射线晶体学、小角 X 射线散射或质谱 (MS)，都需要将膜蛋白完全溶解在水溶液中。去污剂常用于完成这一任务，但可能会干扰分析，就像 MS 那样。使用“MS 友好”去污剂，如非离子烷基糖苷去污剂，极大地促进了去污剂溶解的膜蛋白分析。在这里，我们描述了一种方案，我们已成功地在我们的实验室中实施，以通过氢/氘交换和质谱 (HDX-MS) 研究去污剂溶解的整合膜蛋白的结构和动态。该程序在进行 MS 分析之前不需要去除去污剂，而是利用超高压色谱系统将氘化肽与“MS 友好”去污剂分离。
