Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes
Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes
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DOI：
10.1021/ac101235k
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发表时间：
2010-08-01
影响因子：
7.4
通讯作者：
Yates, John R., III
中科院分区：
文献类型：
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作者：
Fonslow, Bryan R.;Kang, Seong A.;Yates, John R., III
在这里，我们报告使用毛细管等电聚焦在自然条件下分离蛋白质复合物的异构体和亚复合物。使用生物学相关的HIS标签和FLAG标签纯化的蛋白质复合物，我们证明了分离的蛋白质复合物亚型的哺乳动物靶的雷帕霉素复合物（mTORC 1和2）和子复合物和不同的磷酸化状态的Dam1复合物。高效率的毛细管等电聚焦分离允许分辨率的蛋白质复合物和亚复合物相似的大小和生化组成。通过用天然缓冲液和降低的温度（15 ℃）进行分离，我们能够在等电聚焦和检测期间保持更耐热的mTORC 2的复合物完整性（
Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (