Development of Analytical Procedures for the Phosphoproteome Research

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Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells. The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Methods for the determination of the phosphorylation status of a protein are thus very important with respect to the evaluation of the basis for understanding the molecular origins of diseases and for drug design. Recently, we reported that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl) amino]propan-2-olate acts as a phosphate-binding tag molecule, Phos-tag, in an aqueous solution. This finding has contributed to the development of phosphate-selective MS analysis, phosphate-affinity chromatography, phosphate-selective Western analysis, and phosphate-affinity electrophoresis. For example, the phosphate-affinity electrophoresis, Phos-tag SDS-PAGE offers the following significant advantages: i) Radioactive and chemical labels are avoided.ii) The time-course quantitative ratio of the phosphorylated and nonphosphorylated proteins can be determined.iii) The phosphate-binding specificity is independent of the amino acid sequence context. iv) A downstream procedure, such as Western blotting analysis, is applicable. v) The procedure is almost identical to that of the general SDS-PAGE system. We believe that phosphoproteomics would progress greatly by combining our Phos-tag technology and existing methods using high-quality antibodies and convenient mass spectrometers.