Médard Guillaume, Pachl Fiona, Ruprecht Benjamin, Klaeger Susan, Heinzlmeir Stephanie, Helm Dominic, Qiao Huichao, Ku Xin, Wilhelm Mathias, Kuehne Thomas, Wu Zhixiang, Dittmann Antje, Hopf Carsten, Kramer Karl, Kuster Bernhard
Chair of Proteomics and Bioanalytics, Technische Universität München , Freising, Germany.
J Proteome Res. 2015 Mar 6;14(3):1574-86. doi: 10.1021/pr5012608. Epub 2015 Feb 20.
Solid supported probes have proven to be an efficient tool for chemical proteomics. The kinobeads technology features kinase inhibitors covalently attached to Sepharose for affinity enrichment of kinomes from cell or tissue lysates. This technology, combined with quantitative mass spectrometry, is of particular interest for the profiling of kinase inhibitors. It often leads to the identification of new targets for medicinal chemistry campaigns where it allows a two-in-one binding and selectivity assay. The assay can also uncover resistance mechanisms and molecular sources of toxicity. Here we report on the optimization of the kinobead assay resulting in the combination of five chemical probes and four cell lines to cover half the human kinome in a single assay (∼ 260 kinases). We show the utility and large-scale applicability of the new version of kinobeads by reprofiling the small molecule kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil, Nilotinib, Ibrutinib, Imatinib, and Sunitinib.
固相支持探针已被证明是化学蛋白质组学的一种有效工具。激酶磁珠技术的特点是将激酶抑制剂共价连接到琼脂糖上，用于从细胞或组织裂解物中亲和富集激酶组。该技术与定量质谱相结合，对于激酶抑制剂的分析尤为重要。它常常能为药物化学研究确定新的靶点，实现二合一的结合和选择性检测。该检测还能揭示耐药机制和毒性的分子来源。在此，我们报告激酶磁珠检测的优化，通过五种化学探针和四种细胞系的组合，在一次检测中覆盖近一半的人类激酶组（约260种激酶）。我们通过对小分子激酶抑制剂阿沃西地、克唑替尼、达沙替尼、法舒地尔、羟基法舒地尔、尼罗替尼、伊布替尼、伊马替尼和舒尼替尼进行重新分析，展示了新版激酶磁珠的实用性和大规模适用性。
