水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.

Optimization of Two-dimensional Gel Electrophoresis of Plasma Membrane Proteome from Rice Root

A highly purified(90%) plasma membrane fraction was isolated from rice root tips using an aqueous Dextran-Polyethylene Glycol two phase partition system,and solubilized by four different kinds of rehydration.Solubilized plasma membrane fraction was then separated with IEF/SDS-PAGE two-dimensional gel electrophoresis and analyzed by MALDI-TOF/TOF MS.The results showed that most efficient rehydration buffer in this case consisted of 7 mol/L Urea,2 mol/L Thiourea,20 mmol/L DTE,1% amidosulfobetaine-14(ASB14),and 4% 3-(3-Cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate(CHAPS).9 of 16 spots analyzed by MS were the plasma membrane-associated proteins,5 spots were unknown proteins,and only 2 spots represented the contaminated proteins from other organelles.The study indicated that low concentration of ASB-14 is more suitable for the proteomic analysis of the plant plasma membrane.IEF/SDS-PAGE two-dimensional gel electrophoresis in such optimized condition is suitable for separation of hydrophilic peripheral proteins from plasma membrane of rice root.