Two Dimensional Gel Electrophoresis-Based Plant Phosphoproteomics

Abstract

Phosphorylation is one of the most important reversible protein modifications and is involved in regulating signal transduction, subcellular localization and enzyme activity of target proteins. Phosphorylation or dephosphorylation of proteins is directly reflected in changed ratios of phosphoprotein abundance and total protein abundance. Two-dimensional gel electrophoresis (2-DE)-based proteomics allow quantification of both total protein abundance by Coomassie Brilliant Blue (CBB) staining and phosphoprotein abundance by fluorescence-based staining. Pro-Q diamond phosphoprotein stain (Pro-Q DPS) can bind to the phosphate moiety of the phospho-amino acid directly, regardless of the nature of the phospho-amino acid. Phosphoproteins can thus be detected using proper excitation light, quantified using image analysis software and subsequently be subjected to analysis by mass spectrometry. Here, we describe a protein phosphorylation status analysis method combining both CBB and Pro-Q DPS staining based on 2-DE gel-based phosphoproteomics, which has been widely applied to plant phosphoproteomics studies.