Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, approximately 10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

中文翻译：

---

新型广谱激酶抑制剂CTx-0294885的表征，可作为基于质谱的kinome分析的亲和试剂。

利用广谱激酶抑制剂的激酶富集能够通过质谱鉴定大部分表达的激酶组。但是，现有的抑制剂仍然不足以覆盖整个激酶组。在这里，我们确定了一种新型的双氨基吡啶嘧啶CTx-0294885，在体外对多种激酶均表现出抑制活性，并将其进一步开发为琼脂糖支持的激酶捕获试剂。定量蛋白质组学方法的使用证实了结合CTx-0294885的珠子对激酶富集的选择性。基于大规模CTx-0294885的亲和纯化，然后进行LC-MS / MS，可以鉴定出MDA-MB-231细胞中的235种蛋白激酶，包括AKT家族的所有成员，而这些成员以前从未被其他广泛光谱激酶抑制剂。将CTx-0294885添加到通常用于激酶富集的三种激酶抑制剂的混合物中，可使激酶鉴定的数量增加至261，代表迄今为止报道的单个细胞系中最大的激酶组覆盖率。使用这四种抑制剂将磷酸肽富集与亲和纯化相结合，可以鉴定183个激酶上的799个高可信度磷酸位点，其中约10％定位在激活环上，并且包括BMP2K，MELK，HIPK2和PRKDC上以前未报道的磷酸位点。因此，CTx-0294885代表了一种强大的新型试剂，可用于分析激酶组信号网络，从而有助于开发靶向治疗策略。蛋白质组学数据已保存到ProteomeXchange联盟（http：//proteomecentral.proteomexchange。