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.