Shi Chaowei, Han Fang, Xiong Ying, Tian Changlin
National Laboratory for Physical Science at Microscale, School of Life Science, University of Science and Technology of China, 96 Jin Zhai Road, Hefei, Anhui 230026, People's Republic of China.
Protein Expr Purif. 2009 Dec;68(2):221-5. doi: 10.1016/j.pep.2009.07.012. Epub 2009 Jul 28.
In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography-mass spectrometry selected ion monitoring (GC-MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC-MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.
在膜蛋白的生化和结构研究中，去污剂用于模拟膜环境，并在没有脂质双层的情况下维持膜蛋白的功能稳定构象。然而，去污剂浓度，尤其是膜蛋白与去污剂的摩尔比通常是未知的。在此，开发了一种气相色谱 - 质谱选择离子监测（GC-MS-SIM）方法来定量膜蛋白结构研究中常用的四种去污剂。为了去除过量的去污剂，采用了使用Centricon管的过滤离心法。以处于四种不同去污剂胶束中的膜蛋白Ig-β片段为例。在每一轮离心后测量Centricon管上下部分的去污剂浓度。结果与去污剂胶束在水性溶剂中的基本性质非常一致。因此，将GC-MS-SIM与通过Centricon管去除去污剂相结合，可以控制去污剂浓度，尤其是膜蛋白与去污剂的摩尔比，这将加快膜蛋白的结构和生化研究。
