Two-Dimensional Gel Electrophoresis and Pro-Q Diamond Phosphoprotein Stain-Based Plant Phosphoproteomics

Pro-Q diamond phosphoprotein gel stain is a fluorescent stain to detect phosphorylated proteins in polyacrylamide gels with high sensitivity. Here, we describe an entire procedure for phosphoproteomics analysis of Arabidopsis seedlings by a combination of Pro-Q diamond stain and two-dimensional gel electrophoresis (2-DE). The workflow involves total protein preparation, protein separation by 2-DE, the second-dimensional gel staining, phosphoproteins detection, and peptides preparation for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Approximately 300 phosphoproteins can be detected using this method.