Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase–substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase–substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase–substrate pairs are then isolated with high specificity and are identified and quantified by LC–MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by in vitro kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy.

中文翻译：

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荧光互补质谱法鉴定上游激酶

蛋白激酶及其底物包含广泛的信号传导网络，可调节许多不同的细胞功能。但是，系统地鉴定直接引起特定磷酸化事件的激酶的方法和技术仍然难以捉摸。在这里，我们描述了一种称为荧光互补质谱（FCMS）的新型蛋白质组学策略，以高通量鉴定激酶-底物对。FCMS策略采用特定的底物和激酶库，两者均与荧光互补的蛋白片段融合。活细胞中瞬时的和弱的激酶-底物相互作用通过荧光蛋白片段的结合得以稳定。然后以高特异性分离出这些激酶-底物对，并通过LC-MS进行鉴定和定量。FCMS已应用于鉴定转录因子cAMP反应元件结合蛋白（CREB）的已知激酶和新型激酶。新型CREB激酶已通过以下验证在体外激酶测定中，磷酸化位点是明确定位的。这些结果揭示了CREB在多个重要信号通路中可能的新作用，并证明了这种新蛋白质组学策略的巨大潜力。