Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry.
Systematic analysis of the use of amphipathic polymers for studies of outer membrane proteins using mass spectrometry.
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DOI：
10.1016/j.ijms.2015.06.017
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发表时间：
2015-11-30
影响因子：
1.8
通讯作者：
Ashcroft AE
中科院分区：
文献类型：
--
作者：
Watkinson TG;Calabrese AN;Giusti F;Zoonens M;Radford SE;Ashcroft AE
外膜蛋白构象分析。分子筛在质谱分析中的应用。几种分子膜蛋白分析方法的比较。膜蛋白（MP）是许多重要的生物过程所必需的。最近，质谱（MS），加上一系列相关技术，已被用来探测MP及其复合物的结构特性。通常，洗涤剂胶束已被用于将MP递送到气相中，但这些复合物具有固有性质，这可能限制使用MS方法对MP进行结构研究的实用性。Amphipol（APols）具有优于洗涤剂胶束的优点，并且已经显示能够将天然MP递送到气相中。比较六种不同的APOL，其质量和电荷不同，以及去污剂n-十二烷基-β-d-麦芽吡喃糖苷，我们的目的是确定哪些APOL最有效地将天然外膜蛋白（OMP）递送到气相中。我们发现，保持溶液相折叠和全球结构的三种不同的外膜蛋白（PagP，OmpT和tOmpA）是独立的APOL使用，但在OMP活性的差异，可以导致不同的APOL：OMP复合物。OMP：APol复合物的ESI-IMS-MS分析表明，A8-35 APol最擅长将所有三种OMP释放到气相中，而不改变它们的气相构象。
Outer membrane protein conformational analysis. Use of amphipols for mass spectrometry analyses. Comparison of a range of amphipols for membrane protein analysis. Membrane proteins (MPs) are essential for numerous important biological processes. Recently, mass spectrometry (MS), coupled with an array of related techniques, has been used to probe the structural properties of MPs and their complexes. Typically, detergent micelles have been employed for delivering MPs into the gas-phase, but these complexes have intrinsic properties that can limit the utility of structural studies of MPs using MS methods. Amphipols (APols) have advantages over detergent micelles and have been shown to be capable of delivering native MPs into the gas-phase. Comparing six different APols which vary in mass and charge, and the detergent n-dodecyl-β-d-maltopyranoside, we aimed to determine which APols are most efficient for delivery of native outer membrane proteins (OMPs) into the gas-phase. We show that maintaining the solution-phase folding and global structures of three different OMPs (PagP, OmpT and tOmpA) are independent of the APol used, but differences in OMP activity can result from the different APol:OMP complexes. ESI-IMS–MS analysis of OMP:APol complexes shows that the A8-35 APol is most proficient at liberating all three OMPs into the gas-phase, without altering their gas-phase conformations.