Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).
Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).
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DOI：
10.3389/fphys.2015.00098
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发表时间：
2015
影响因子：
4
通讯作者：
Yan LJ
中科院分区：
文献类型：
--
作者：
Wu J;Luo X;Yan LJ
脂质过氧化产物4-羟基壬烯醛（HNE）可以形成蛋白质连接的HNE加合物，从而影响蛋白质的结构和功能。线粒体复合物I（NADH-泛醌氧化还原酶），在哺乳动物细胞中含有至少45个亚基，位于富含脂质的环境中，因此对HNE修饰非常敏感。在本文中，HNE修饰的复合物I亚基的鉴定程序。复合物I通过第一维非梯度蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）分离。通过考马斯蓝染色或银染色观察分离的复合物I带，通过第二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进一步分析。HNE修饰的蛋白质通过用抗HNE抗体探测的蛋白质印迹法可视化。然后切下HNE阳性条带，并通过质谱肽测序鉴定其中所含的蛋白质。该方法被成功地应用于两个复杂的I亚基，显示增强HNE修饰糖尿病肾线粒体的鉴定。
The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.
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