Development Of 2-DE Protocol For Suitable Wheat Leaf Proteome Analysis

Sample preparation is one of the key factors which determine resolution and reproducibility of two-dimensional electrophoresis. In plant proteomics,extraction of total protin is much more complex than animal and micro-organism's because plant tissues have their own features. At present, plant total protein is usually extracted with TCA/acetone method,but the researchers find that protein extracted is difficult to dissolve and loss of protein is more .To develop sample preparation methods suitable for wheat leaf proteome analysis in future is pressing. Using wheat vatiety Taichung29 ,the study first select suitable extraction method through SDS-PAGE from three extraction methds :TCA/acetone method, phenol extraction-methanol/ammonium acetate precipitation and urea/thiourea extraction,then utilizes the two methods which loss of protein is few compared with TCA/acetone method to extract wheat leaf protein. The total protein is separated by two-dimensional electrophoresis, followed by colloidal Coomassie Blue G-250 staining. Some protein spots were selected for MALDI-TOF MS analysis and database searching to analysis differences between the selected methods. All of this is for comparative proteomics research on analysis of stripe rust resistance in wheat.From the SDS-PAGE gel,there are some differences in protein location ,content and the number of protein. Using the selected two methods: phenol extraction-methanol/ammonium acetate precipitation and urea/thiourea extraction, proein mixture is separated through IEF which uses pH3 -10 and pH4-7 IPG strip and SDS-PAGE in the first and second dimension respectively, 1016±203, 1014±281 and 1563±37, 1555±280 spots could be detected respectively on 2-DE gels, and the selected protein spots could be efficiently identified. In the scope of pH 3-10 , basic proteins can be detected through phenol extraction-methanol/ammonium acetate precipitation; urea/thiourea extraction is profit for protein separation which molecular mass is above 70kDa. In the scope of pH 4-7, urea/thiourea extraction can easily separate acid proteins,furthermore can relatively dissolve much more protein compared with phenol extraction-methanol/ammonium acetate precipitation. In the course of peptide mass fingerprinting analysis,baseline of protein spectrum map is steady so the whole protocol is comparable with mass spectrum.The two sample preparation methods provided here which can sepatate mssive protein spots and be comparable with mass spectrum could be used in wheat leaf proteome analysis in future.