水稻根尖质膜蛋白质组学研究方法的建立

 以徐稻3号水稻苗期幼嫩根尖为材料，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，使用优化的双向电泳水化液溶解质膜蛋白，通过等电聚焦/十二烷基磺酸钠 聚丙烯酰胺凝胶双向电泳 （IEF/SDS PAGE）分离和基质辅助激光解吸电离串联飞行时间质谱（MALDI TOF/TOF）分析，鉴定了31个水稻质膜相关蛋白。结果表明IEF/SDS PAGE 双向电泳适合分离亲水性相对较高的膜附着蛋白。进一步利用高盐和温和去污剂对纯化的质膜进行洗涤，以降低质膜组分的复杂程度，通过 SDS PAGE 单向电泳分离和液相色谱串联质谱（LC MS/MS）分析，鉴定了 8个质膜蛋白。经洗涤后的质膜组分复杂度显著降低，SDS PAGE 中的蛋白条带只包含1~2 种蛋白，且主要为疏水性较强的穿膜蛋白，说明多种生物化学分离方法及不同质谱分析的综合运用，是解决生物膜蛋白质组学难点的有效途径。

Establishment of Technique for Plasma Membrane Proteomics from Rice Root Tips

A highly purified plasma membrane fraction was isolated from rice root tips using an aqueous two-polymer phase-partition system,and the proteins in the fraction were solubilized by optimized rehydration buffer.Solubilized plasma membrane fraction was then separated with two dimensional gel electrophoresis.Among the result,31 membrane-associated proteins,visualized clearly,were analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF/TOF) mass spectrum.It indicated that isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis(IEF/SDS-PAGE) was fit for the separation of hydrophilic peripheral proteins of the plasma membrane.In order to reduce the complexity of the fraction,high-salt solutions and gentle detergents had been used.According to the result of the SDS-PAGE and nanoelectrospray ionization tandem mass spectrometry(ESI-MS/MS),eight integral proteins which contained several transmembrane domains had been identified.It suggested that the complexity of the purified membrane was reduced.Each strip of the SDS-PAGE contained one or two proteins.Taken together,the integration of various separation methods and different mass spectrums analysis would be the validity means for the plasma membrane proteomics.The optimization of the membrane proteomics method paved the way for the comparative proteomic analysis of rice plasma membrane under stresses.