Use of a modified Multiplexed Inhibitor Bead assay to study kinome responses to cytostatic treatment in human cancer cell lines Triplicate Test: pilot study

In order to improve elution of proteins from the column, to reduce the cost as well as the hands on time of the analysis, we here present a modification of the Multiplexed Inhibitor Bead assay (MIB assay), where we elute the proteins from beads containing covalently bound inhibitors using trypsin digestion. Three human cancer cell lines treated with the same chemotherapeutics, cisplatin and docetaxel, were used to monitor and evaluate the performance of the assay. Using this method, we see a good coverage of the kinome as well as other important signaling proteins using only three kinase inhibitors. We demonstrate that the method is reproducible and that the column is re-usable for more than 10 times without loss in performance. The MIB assay revealed large differences in the cellular signaling responses between the cell lines.

为提升蛋白质从层析柱中的洗脱效率、降低分析成本与实操时长，本研究对多重抑制剂微球检测法（Multiplexed Inhibitor Bead assay，MIB assay）进行了改良，采用胰蛋白酶消化法从共价结合抑制剂的微球上洗脱靶蛋白。我们选用经相同化疗药物顺铂（cisplatin）与多西他赛（docetaxel）处理的三株人癌细胞系，对该检测方法的性能开展监测与评估。借助该方法，仅使用三种激酶抑制剂，即可实现激酶组（kinome）及其他重要信号蛋白的高效覆盖。研究证实，本方法具备良好重现性，且层析柱可重复使用10次以上而性能无衰减。该MIB检测法揭示了不同细胞系间细胞信号应答的显著差异。