Development of Analytical Procedures for the Phosphoproteome Research

蛋白质磷酸化对于所有活细胞中的生物过程的调节事件（例如信号转导、凋亡、增殖、分化和代谢）是必需的。激酶和磷酸酶反应的平衡控制着某种蛋白质的磷酸化状态。平衡的扰动触发严重的病理，如癌症和炎症。因此，测定蛋白质磷酸化状态的方法对于理解疾病的分子起源和药物设计的基础的评价是非常重要的。最近，我们报道了1，3-双[双（吡啶-2-基甲基）氨基]丙-2-醇的双核金属配合物在水溶液中作为磷酸盐结合标签分子Phos-tag。这一发现促进了磷酸盐选择性MS分析、磷酸盐亲和色谱、磷酸盐选择性Western分析和磷酸盐亲和电泳的发展。例如，磷酸盐亲和电泳，Phos标签SDS-PAGE提供了以下显著的优点：i）避免了放射性和化学标记; ii）可以确定磷酸化和非磷酸化蛋白质的时间过程定量比率; iii）磷酸盐结合特异性与氨基酸序列背景无关。iv）下游程序，如蛋白质印迹分析，是适用的。v）程序与一般SDS-PAGE系统的程序几乎相同。我们相信，通过将我们的Phos标签技术与使用高质量抗体和方便的质谱仪的现有方法相结合，磷酸化蛋白质组学将取得巨大进展。

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.