Activation and Regulation of the Understudied Kinome Using MIB/MS Technology

描述：已经实施了一种新的方法来研究蛋白激酶的整体激活状态。我们的方法允许从细胞系、组织和肿瘤中分离和分析蛋白激酶，这些蛋白激酶在使用多重抑制物珠(MIB)的单次质谱分析中得到分析。MIBs由琼脂糖珠和共价固定化、连接子适应的激酶抑制剂的混合物组成。激酶捕获是可重复性的，是以下各项的函数 酶对不同固定化抑制剂的亲和力以及酶的激活状态 激活剂。我们合成的用于连接琼脂糖珠的抑制剂主要是1型抑制剂，它优先结合蛋白激酶的激活状态。广泛性 对MIB的表征表明，它们有很强的结合活性状态的激酶的偏好，而非活性的激酶结合得很差。使用MIB柱的分层珠子成分，我们能够在一次质谱分析中捕获和识别超过275个激酶。根据RNA-seq分析，MIB至少捕获了细胞系、肿瘤或正常组织中表达的动态组的75%-80%。重要的是，我们已经从细胞和组织裂解物中捕获并表征了多达275个蛋白激酶，其蛋白质含量低至500毫克。当组织受到限制时，这种程度的灵敏度对于动态组分析是必不可少的。因此，MIB/MS提供了一种无与伦比的、可扩展的、基于生物学的高通量技术来分析包括“非靶向和未被研究的”激酶在内的动态组的激活状态。对于许多“未被研究的”激酶，很少或根本没有试剂，从而限制了它们的研究，而MIB/MS克服了这些限制。MIB/MS还检测试剂无法区分密切相关的激酶的活性变化(例如，MEK1与MEK2和ERK1与ERK2的不同活性状态，它们是通过无法区分异构体的抗磷抗体一起评估的)。同样，我们可以很容易地区分相关受体酪氨酸激酶(RTK)的调节，如DDR1和DDR2，而对它们的研究试剂很少。AIMS将定义全面的动态组激活签名，其中包括糟糕的或未刻画的激酶。目的包括：1.利用MIB/MS对跨越不同癌症类型的一系列人类细胞系、人和小鼠肿瘤以及小鼠正常组织进行Key组激活状态的测定。2.使用针对蛋白质和脂蛋白激酶、细胞骨架、代谢调节剂、DNA损伤和表观遗传染色质修饰酶的特定化学探针来确定动态组对细胞生理扰动的反应。3.确定未被研究的激酶的RNAi敲除对激动组激活状态的影响。这些目的强调确定未被研究的激酶的激活状态，以及特定的未被研究的激酶是否作为响应特定细胞扰动的激活/抑制信号的一部分发挥作用。这一分析将确定未被研究的激酶活性受到调控的信号网络和细胞功能，并有助于内环境平衡控制机制。我们的目标是确定和验证哪些目前未被研究的激酶作为Kinome内的调节节点，并保证未来的化学探针开发。

DESCRIPTION: A novel approach has been implemented to study the activation state of protein kinases "en masse". Our methods allow isolation and analysis of protein kinases from cell lines, tissues and tumors assayed in a single mass spectrometry run using Multiplexed Inhibitor Beads (MIBs). MIBs consist of mixtures of Sepharose beads with covalently immobilized, linker adapted, kinase inhibitors. Kinase capture is reproducible and is a function of affinity of kinases for the different immobilized inhibitors as well as the activation state of the kinase. The inhibitors we have synthesized for linker conjugation to Sepharose beads are primarily type 1 inhibitors that preferentially bind activated states of protein kinases. Extensive characterization of the MIBs has demonstrated they have a strong preference for binding the active state of kinases and inactive kinases bind poorly. Using a layered bead composition for MIB columns, we are able to capture and identify more than 275 kinases in a single mass spectrometry run. Based on RNA-seq analysis, MIBs are capturing at least 75-80% of the expressed kinome from cell lines, tumors or normal tissues. Importantly, we have captured and characterized up to 275 kinases with as little as 500 mg protein from cell and tissue lysates. This level of sensitivity is essential for kinome analysis when tissue is limiting. Thus, MIB/MS provides an unparalleled scalable biology- based high throughput technology to assay the activation state of the kinome including "untargeted and understudied" kinases. There simply are few or no reagents for many of the "understudied" kinases, thereby limiting their study and MIB/MS overcomes these limitations. MIB/MS also detects activity changes in kinases for which reagents cannot distinguish closely related kinases (e.g., different activity states of MEK1 vs. MEK2 and ERK1 vs. ERK2, which are assessed together by anti-phospho-antibodies that cannot distinguish isoforms). Similarly, we can easily distinguish the regulation of related receptor tyrosine kinases (RTKs), such as DDR1 and DDR2, for which there are few reagents for study. The aims will define comprehensive kinome activation signatures that include poorly or uncharacterized kinases. Aims include: 1. Determine the activation state of the kinome using MIB/MS for a series of human cell lines spanning different cancer types, human and mouse tumors, and normal tissues of the mouse. 2. Determine the response of the kinome to perturbation of cellular physiology using specific chemical probes targeting protein and lipid kinases, cytoskeleton, metabolic regulators, DNA damage and epigenetic chromatin modifying enzymes. 3. Determine the consequence of RNAi knockdown of understudied kinases on the activation state of the kinome. The aims emphasize determining the activation state of understudied kinases and if specific understudied kinases function as part of a signature of kinase activation/inhibition in response to specific cellular perturbations. This analysis will defne the signaling networks and cellular functions for which the activity of understudied kinases are regulated and contribute to homeostatic control mechanisms. The goal is to identify and validate which current understudied kinases function as regulatory nodes within the kinome and warrant future chemical probe development.