膜タンパク質をターゲットとしたプロテオーム解析基盤技術の確立

抄録

[Introduction] Two-dimensional gel electrophoresis is a powerful technique enabling visualization of the proteome. Recently, it is reported that fluorescence labeled two-dimensional gel electrophoresis is one of the most powerful method for comprehensive proteomic analysis. While annotated two-dimensional gel electrophoresis contain thousands of proteins, they do not represent the entire genome. Hydrophobic membrane proteins in particular are conspicuously absent from such data. There are a lot of membrane proteins with important physiological functions that is involved in signal transduction etc. Recently, the improvement of the solubilize conditions for these membrane proteins are required. Therefore, we examine the application of several non-ionic detergent for two-dimensional gel electrophoresis. [Methods] Microsomal membrane fraction was obtained by centrifugation of rat brain homogenates. Membrane fraction was solubilized with sample buffer including different detergent. Two-dimensional gel electrophoresis, isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, was performed using IEF Cell (Bio-Rad). [Results] We examined some detergents and non-detergent sulfobetaines, and compared them with CHAPS that is generally used in two-dimensional gel electrophoresis. As a result, it was suggested that some non-ionic detergents improved separation in particular high molecular weight protein region. We were able to identify some membrane proteins by peptide mass fingerprinting methods using MALDI-TOF/TOF MS. This study provides methodological tools to study particular classes of membrane proteins and should be applicable to other cellular membranes such as raft microdomain.