Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment
Measurement of the molecular masses of hydrophilic and hydrophobic subunits of ATP synthase and complex I in a single experiment
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DOI：
10.1016/j.ab.2009.08.006
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发表时间：
2009-12-15
影响因子：
2.9
通讯作者：
Walker, John E.
中科院分区：
文献类型：
--
作者：
Carroll, Joe;Fearnley, Ian M.;Walker, John E.
线粒体中的三磷酸腺苷（ATP）合酶和复合物I是亲水和疏水蛋白质的膜结合多亚基组装体。然而，它们的分子量的质谱测量需要在两个不同的实验中将许多疏水蛋白与其他组分分开分析。在这里，我们描述了一个程序，允许所有的分子量，或几乎所有的，每个复杂的亚基在一个单一的实验中进行测量。关键特征是移动的相，亲水和疏水组分在其中保持可溶性，这与反相色谱兼容。以这种方式，测量了牛ATP合酶的所有17个亚基、来自酿酒酵母的酶的17个亚基中的14个、牛复合物1的45个亚基中的42个以及牛亚复合物批次的所有28个亚基的质量。该方法被用来表征亚基的ATP酶和复合物I从各种物种，并遵循温和的胰蛋白酶ATP合酶的进展。它可以应用于其它呼吸和光合复合物，并且通常应用于含有亲水和疏水亚基的任何蛋白质复合物。(C)2009 Elsevier Inc. All rights reserved.
The adenosine triphosphate (ATP) synthase and complex I in mitochondria are membrane-bound multi-subunit assemblies of both hydrophilic and hydrophobic proteins. Hitherto, the mass spectrometric measurement of their molecular masses has required that many of the hydrophobic proteins be analyzed separately from the other components in two different experiments. Here we describe a procedure that allows the molecular masses of all, or nearly all, of the subunits of each complex to be measured in a single experiment. The key feature is a mobile phase, in which hydrophilic and hydrophobic components remain soluble, that is compatible with reverse phase chromatography. In this way, the masses of all 17 subunits of bovine ATP synthase, 14 of the 17 subunits of the enzyme from Saccharomyces cerevisiae, 42 of the 45 subunits of bovine complex 1, and all 28 of the subunits of bovine subcomplex lot were measured. The method was used to characterize the subunits of ATP synthases and complexes I from a variety of species and to follow the progress of mild trypsinolysis of ATP synthase. it could be applied to other respiratory and photosynthetic complexes and, in general, to any protein complex that contains both hydrophilic and hydrophobic Subunits. (C) 2009 Elsevier Inc. All rights reserved.