The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay
The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay
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DOI：
10.1074/mcp.t600027-mcp200
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发表时间：
2006-09-01
影响因子：
7
通讯作者：
McDowall, Kenneth J.
中科院分区：
文献类型：
--
作者：
Stead, Jonathan A.;Keen, Jeff N.;McDowall, Kenneth J.
与核酸相互作用的蛋白质是许多细胞过程的核心，它们的持续表征代表了后基因组时代最重要的挑战之一。在这里，我们描述了一种简单的基于蛋白质组学的方法，通过质谱法鉴定与核酸相互作用的粗提取物中的蛋白质。它结合了电泳迁移率转移测定，并基于这样的发现：当蛋白质与核酸形成复合体时，其电泳迁移率和核酸的迁移率一样受到影响。我们的方法应该大大减少，在某些情况下甚至可能消除对大量蛋白质纯化的需要，因此应该对蛋白质组的功能注释做出重大贡献。此外，它不需要事先了解相互作用蛋白质的分子质量、四级结构或pI。使用最近发现的转录因子来证明原理；然而，这种方法也应该应用于鉴定与RNA相互作用的蛋白质。
Proteins that interact with nucleic acids are central to numerous cellular processes, and their continuing characterization represents one of the foremost challenges in the postgenomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.