Comparative analysis of phosphoprotein expression using 2D-DIGE.

Two-dimensional gel electrophoresis (2-DE) has often been used to compare protein expression pattern between different samples. This method is also useful to compare protein expression between wild-type and RNAi plants. 2D-DIGE (difference gel electrophoresis) was developed to perform quantitative proteomics of two or more samples. In this method, proteins are fluorescently labeled by Cy2, Cy3, or Cy5 and are mixed and subjected to electrophoresis on the same gel, thereby gel-to-gel variations are eliminated. We perform phosphoproteomics between Arabidopsis wild-type and a stress-activated MAPK (mitogen-activated protein kinase) mutant after the phytotoxin treatment. For this purpose, proteins are extracted from both samples, and then phosphorylated proteins are purified by phosphoprotein enrichment columns. Purified proteins are fluorescently labeled by different CyDyes using the minimum labeling method. The labeled protein samples are separated on the same gel by 2-DE, and gels are scanned by a variable mode laser imager. Then, high-resolution images are analyzed by 2D analysis software. Thus, we identified several phosphoprotein spots that were differentially accumulated between wild-type and mapk mutant.