Kinase-centric chemoproteomics utilizing targeted affinity enrichment and state-of-the-art quantitative mass spectrometry analyses

蛋白质磷酸化激酶作为一个重要的分子开关在大多数细胞过程。激酶激活和关闭信号级联，介导蛋白质-蛋白质相互作用，它们的失调通常导致癌症等疾病。因为它们作为细胞刺激的整合子和效应子，所以激酶的丰度和活性经常被用作细胞状态的报告子。激酶抑制剂最近已被用作亲和剂与定量质谱（MS）分析，以概况激酶抑制剂的选择性。虽然证明了富集激酶用于全局蛋白质组学分析的能力，但这些研究没有在功能上询问特定的信号传导途径或激酶的活化状态。在这里，我们提出了一种策略，利用有针对性的亲和富集和定量质谱分析，使在深入表征丰富的激酶和相应的信号传导模块的功能状态。为了实现以激酶为中心的蛋白富集，将制备两个系列的基于小分子（SM）激酶通道的亲和剂。第一系列包括I型激酶抑制剂类似物，其允许选择性或全局的、不依赖于激活状态的激酶及其相互作用伴侣的富集。第二系列由II型激酶抑制剂组成，其促进激酶和相关蛋白的活化状态依赖性富集。在概念验证研究中，由这些抑制剂类似物制备的亲和基质将用于参与表皮生长因子（EGFR）-促分裂原活化蛋白激酶（MAPK）信号传导的激酶和相关细胞组分的途径特异性和活化状态依赖性富集。七种不同的NCI 60细胞系类似于重要的人类癌症病理将作为模型蛋白质组。将通过定量MS分析富集的亚蛋白质组，包括（i）EGFR-MAPK信号传导组分的富集效率，（ii）鉴定和定量这些组分的准确性和重现性，以及（iii）表征富集的激酶和相关蛋白质（包括翻译后修饰（PTM））的能力。这将有助于EGFR-MAPK信号传导模块的功能表征。在稍后的阶段，我们希望将我们的方法应用于来自患者样本的高级ErbB信号网络和蛋白质组。我们的长期目标是为功能性癌症蛋白质组学提供新的分析工具，使异常激酶信号转导的快速和深入表征成为可能。所获得的蛋白质组水平的信息应该补充从癌症基因组测序中获得的基因组水平的信息，从而扩展了对抗癌症的分析军械库。

Protein phosphorylation by kinases serves as an important molecular switch in most cellular processes. Kinases activate and shut down signaling cascades, mediate protein-protein interactions and their dysregulation commonly leads to diseases like cancer. Because they act as integrators and effectors of cellular stimuli, the abundance and activity of kinases are often used as reporters for the cellular state. Kinase inhibitors have recently been used as affinity agents together with quantitative mass spectrometry (MS) analyses to profile kinase inhibitor selectivity. While demonstrating the ability to enrich kinases for global proteomic analyses, these studies did not functionally interrogate specific signaling pathways or the activation states of kinases. Here we suggest a strategy utilizing targeted affinity enrichment and quantitative mass spectrometry analyses enabling the in depth characterization of abundance of kinases and the functional state of the corresponding signaling modules. To achieve kinase-centric protein enrichment, two series of small molecule (SM) kinase inhibitor-based affinity agents will be prepared. The first series comprises type I kinase inhibitor analogs allowing selective or global, activation-state independent, enrichment of kinases and their interaction partners. The second series consists of type II kinase inhibitors facilitating activation state-dependant enrichment of kinases and associated proteins. In a proof of concept study, affinity matrices prepared from these inhibitor analogs will be used for the pathway-specific and activation state-dependent enrichment of kinases and associated cellular components involved in epidermal growth factor (EGFR) - mitogen activated protein kinase (MAPK) signaling. Seven different NCI60 cell lines resembling important human cancer pathologies will serve as the model proteomes. The enriched sub-proteomes will be analyzed by quantitative MS in regard to (i) enrichment efficiency of EGFR - MAPK signaling components, (ii) accuracy and reproducibility of the identification and quantitation of these components and (iii) the ability to characterize the enriched kinases and associated proteins, including post-translational modifications (PTMs). This should facilitate the functional characterization of the EGFR - MAPK signaling module. On a later stage, we wish to apply our approach to the superordinate ErbB signaling network and proteomes derived from patient samples. Our long term goal is to provide novel analytical tools for functional cancer proteomics enabling rapid and in depth characterization of aberrant kinase signaling. The proteome-level information obtained should complement genome-level information obtained from sequencing cancer genomes and thus extend the analytical armory for the fight against cancer.