Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli.
Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli.
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DOI：
10.1021/acs.jproteome.7b00777
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发表时间：
2018-02
影响因子：
4.4
通讯作者：
Mamiyo Kawakami;N. Juge;Yuri Kato;H. Omote;Y. Moriyama;T. Miyaji
中科院分区：
文献类型：
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作者：
Mamiyo Kawakami;N. Juge;Yuri Kato;H. Omote;Y. Moriyama;T. Miyaji
纯化的活性膜蛋白的结构分析可以通过质谱法（MS）进行。然而，活性真核细胞膜蛋白的大规模表达系统是可用的。此外，由于膜蛋白不能很容易地被胰蛋白酶消化和离子化，它们很难通过MS分析。我们开发了一种方法，结合在大肠杆菌中的过表达系统的真核细胞膜蛋白的质谱分析。将N端和C端分别带有可溶性α-螺旋蛋白和组氨酸标签的囊泡谷氨酸转运蛋白2（VGLUT 2/SLC 17 A6）在E.大肠杆菌，用去污剂溶解，并通过Ni-NTA亲和层析纯化。含有VGLUT 2的蛋白脂质体保留谷氨酸转运活性。对于MS分析，通过三氯乙酸沉淀从纯化的VGLUT 2中除去去污剂，然后对VGLUT 2进行还原性烷基化和胰蛋白酶消化。通过基质辅助激光解吸电离飞行时间（MALDI-TOF）MS（有或没有液相色谱法）检测所得肽，覆盖率为88%。囊泡兴奋性氨基酸转运体和囊泡乙酰胆碱转运体也用相同的方法检测到相似的覆盖率。因此，该方法可用于分析纯化的真核生物主动转运蛋白。MS化学修饰剂的结构分析可以应用于药物发现的功能结合分析。
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.