Reifschneider Nicole H, Goto Sataro, Nakamoto Hideko, Takahashi Ryoya, Sugawa Michiru, Dencher Norbert A, Krause Frank
Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany.
J Proteome Res. 2006 May;5(5):1117-32. doi: 10.1021/pr0504440.
In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.
根据其多种功能，线粒体的各种功能障碍与大量疾病及衰老过程密切相关。这激发了人们通过变性方法来鉴定所有线粒体蛋白质的大量努力，尤其是采用等电聚焦的标准基于凝胶的方法。由于线粒体蛋白质组的很大一部分与膜相关，并且/或者作为同聚体或异聚体蛋白质复合物发挥作用，因此迫切需要在保留蛋白质 - 蛋白质相互作用的条件下检测和鉴定线粒体蛋白质，包括膜结合蛋白和可溶性蛋白。在这里，我们研究了用洋地黄皂苷溶解的五种不同大鼠器官（肾脏、肝脏、心脏、骨骼肌和大脑）的线粒体，从而能够定量提取五种氧化磷酸化（OXPHOS）复合物。通过蓝色天然（BN）-PAGE分析，OXPHOS复合物在很大程度上以超复合物形式得到恢复，并分离出许多其他蛋白质复合物和单个蛋白质，随后通过二维SDS-PAGE对这些蛋白质进行解析，揭示了组织特异性的线粒体蛋白质组。使用质谱肽质量指纹图谱，我们在所有五个器官中鉴定出92种非冗余的可溶性和膜嵌入非OXPHOS蛋白质，其中许多是已知线粒体蛋白质复合物的组成成分，以及一些新的复合物，例如推定的表观质量约为1800 kDa的“类stomatin蛋白2复合物”。有趣的是，鉴定列表中包括36种已知或推测定位于非线粒体区室的蛋白质，例如糖酵解酶、网格蛋白重链、含缬酪肽蛋白/p97、VoV1-ATP酶和钠钾ATP酶。我们预计，可以鉴定出通过二维BN/SDS-PAGE分离的、代表部分“蛋白质相互作用组”图谱的、洋地黄皂苷溶解的大鼠线粒体中200多种不同的非OXPHOS蛋白质。
