Department of Biochemistry and Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2011 Apr 1;83(7):2767-74. doi: 10.1021/ac2000708. Epub 2011 Mar 11.
Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high-throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO, that is multifunctionalized with titanium(IV) ions for specific binding to phosphoproteins and with biotin groups that allow for enzyme-linked spectrometric detection. The sensitivity, specificity, and quantitative nature of pIMAGO for phosphorylation assays were examined with standard phosphoproteins and with purified phosphoproteins from whole cell extracts. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.
定量磷酸化分析对于理解细胞信号转导至关重要。在这里，我们介绍了一种新的技术，用于在高通量格式下高效测定蛋白质磷酸化，而无需使用磷酸特异性抗体。该技术基于一种水溶性纳米级聚合物，称为 pIMAGO，它多功能化钛（IV）离子，用于特异性结合磷酸化蛋白，以及生物素基团，允许酶联光谱检测。使用标准磷酸化蛋白和来自全细胞提取物的纯化磷酸化蛋白检查了 pIMAGO 用于磷酸化测定的灵敏度、特异性和定量性质。使用 pIMAGO 化学发光测定法可以定量测量低至 100 pg 的磷酸化蛋白。该 pIMAGO 测定法已应用于体外激酶测定、激酶抑制剂筛选和内源性磷酸化事件的测量。该技术提供了一种通用的、定量的方法，用于具有高灵敏度和特异性的全局磷酸化分析。
