Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

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大肠杆菌中过量表达的主动转运蛋白的高效质谱分析

纯化的活性膜蛋白的结构分析可以通过质谱（MS）进行。但是，没有用于活性真核膜蛋白的大规模表达系统。此外，由于膜蛋白不易被胰蛋白酶消化和离子化，因此很难通过质谱分析。我们开发了一种结合大肠杆菌中过表达系统的真核膜蛋白质谱分析方法。分别在N和C末端具有可溶性α螺旋蛋白和组氨酸标签的囊泡谷氨酸转运蛋白2（VGLUT2 / SLC17A6）在大肠杆菌中过表达，用去污剂溶解，并通过Ni-NTA亲和色谱纯化。含有VGLUT2的蛋白脂质体保留了谷氨酸的转运活性。对于MS分析，通过三氯乙酸沉淀从纯化的VGLUT2中除去去污剂，然后对VGLUT2进行还原烷基化和胰蛋白酶消化。使用或不使用液相色谱，通过基质辅助激光解吸电离飞行时间（MALDI-TOF）MS检测得到的肽具有88％的覆盖率。用相同的方法也检测到了囊泡兴奋性氨基酸转运蛋白和囊泡乙酰胆碱转运蛋白。因此，该方法可用于分析纯化的真核活性转运蛋白。MS使用化学修饰剂进行结构分析可用于药物发现的功能结合分析。