McDonald Todd, Sheng Simon, Stanley Brian, Chen Dawn, Ko Young, Cole Robert N, Pedersen Peter, Van Eyk Jennifer E
Department of Medicine, The Technical Implementation and Coordination Core of The Johns Hopkins NHLBI Proteomics Center, The Johns Hopkins University, Baltimore, MD 21224, USA.
Mol Cell Proteomics. 2006 Dec;5(12):2392-411. doi: 10.1074/mcp.T500036-MCP200. Epub 2006 Sep 25.
Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the alpha-subunit of F1F0 ATP synthase that differed due to a change in pI or hydrophobicity.
目前，尚无单一的蛋白质组学技术具备足够的分析能力来检测细胞器、细胞或组织的完整蛋白质组。一种可用于扩大蛋白质组覆盖范围的方法是使用多种分离技术，特别是当不同方法所观察到的蛋白质重叠最少时。以线粒体内膜亚蛋白质组作为模型蛋白质组，我们首次比较了三种蛋白质分离方法（使用贝克曼库尔特公司的ProteomeLab PF 2D蛋白质分级系统进行二维液相色谱、一维反相高效液相色谱和二维凝胶电泳）在确定这些技术的蛋白质分离相对重叠方面的能力。来自这些不同方法的数据表明，重叠的蛋白质数量极低，任意两种技术之间共同的蛋白质不到24%，而三种方法共同的蛋白质仅为7%。利用这三种技术创建了一个总计348个非冗余蛋白质的复合数据库。这些蛋白质中有82%在该亚蛋白质组的蛋白质组学研究中此前未被观察到，而44%在完整线粒体的蛋白质组学研究中未被鉴定。发现每种蛋白质分离方法都成功解析了一个独特的蛋白质子集，液相色谱方法更适合分析跨膜结构域蛋白质和发现新蛋白质。我们还证明，一维和二维液相色谱都能分离F1F0 ATP合酶的α亚基，该亚基因pI或疏水性的变化而有所不同。
