Protein phosphorylation is one of the most frequently occurred posttranslational modifications and plays a critical role in cellular regulatory events. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine and tyrosine residues. In fact, phosphorylation of proteins plays a key role in oncogenesis, cell signaling, apoptosis and immune disorders1. Despite the importance and widespread occurrence of this modification, profiling of phosphoproteins in cells is still a challenge, due to the low copy of phosphorylated proteins in cell and the relative amount of phosphoproteins compared to unphosphorylated proteins.
Radiolabeling by 32P labeling is frequently used conventional method for investigation of phosphoprotein profile in conjunction with 2-DE or 1-D gel electrophoresis and autoradiogram. Alternatively, western blot analysis probed by phosphoprotein-specific antibody is also used for this purpose.
Mass spectrometry has been shown to be a reliable and routine tool to identify proteins in a high throughput manner. However, the identification of phosphorylation by mass spectrometry is not a trivial matter and to this day is not routine also due to the low copy of phosphorylated proteins in cells.
This phosphoprotein enrichment and exclusion of unphosphorylated proteins provides advanced chance in detecting protein phosphorylation in gels with non-radiolabeling method(eg. Staining with fluorescence dye) and enables quantitative comparison between cells.