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发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。
A parallel method for the screening and selective evaluation of inhibitors of multiple cellular endogenous kinases based on capillary electrophoresis (CE) - laser induced fluorescence (LIF) detection was developed. The efficient separation capability of CE and the high selectivity of LIF detectors make it possible to test the activity of multiple intracellular kinases simultaneously. Four cell lines, three specific protein kinase substrate peptides, two selective protein kinase inhibitors and one non-selective protein kinase inhibitor were used in the method. Specific substrate peptides were incubated with cell lysates, selectively phosphorylated by their corresponding kinases, and phosphorylated products and substrate peptides were detected by CE-LIF. Simultaneous determination of the inhibitory activity of one inhibitor against several protein kinases was used to evaluate the selectivity of the inhibitor. This multi-target cell lysate-based screening method provides more information and is more efficient than the traditional single-target screening mode, and the cell lysis solution greatly reduces screening costs as a cheap source of kinase.