Sun Hongyan, Lu Candy H S, Shi Haibin, Gao Liqian, Yao Shao Q
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.
Nat Protoc. 2008;3(9):1485-93. doi: 10.1038/nprot.2008.139.
Protein phosphorylation and dephosphorylation play an important role in regulation of intracellular signal transduction pathways in the biological system. A key step in the biological characterization of phosphatases and their use as drug targets is the identification of their cellular partners and suitable substrates for potential inhibitor development. Herein we describe a microarray-based protocol to map the substrate specificity of protein Ser/Thr phosphatases. This protocol uses Pro-Q dye to sensitively and quantitatively detect the amount of dephosphorylation that occurs from many putative peptide substrates in parallel, and therefore could be used to generate the so-called peptide substrate fingerprints as well as detailed kinetic information of a target phosphatase. Excluding the synthesis of the peptide substrates, the whole protocol takes a total of 11 h to complete and in future can be readily extended to the study of other classes of phosphatases, i.e., protein tyrosine phosphatases.
蛋白质磷酸化和去磷酸化在生物系统中细胞内信号转导通路的调节中发挥着重要作用。磷酸酶生物学特性鉴定及其作为药物靶点应用的关键步骤是确定其细胞内相互作用分子以及用于潜在抑制剂开发的合适底物。在此，我们描述了一种基于微阵列的方法来绘制蛋白质丝氨酸/苏氨酸磷酸酶的底物特异性图谱。该方法使用Pro-Q染料灵敏且定量地并行检测许多假定肽底物发生的去磷酸化量，因此可用于生成所谓的肽底物指纹图谱以及目标磷酸酶的详细动力学信息。除了肽底物的合成外，整个实验方案总共需要11小时完成，并且未来可以很容易地扩展到其他类型磷酸酶的研究，即蛋白质酪氨酸磷酸酶。
