Ultrasensitive pathway phosphoproteomics

描述（由申请人提供）：位点特异性蛋白磷酸化对哺乳动物细胞的信号转导至关重要，磷酸化蛋白水平的扰动是癌变的标志。不幸的是，没有灵敏的方法可以同时检测多个磷酸化事件，因此研究人员被迫进行多次Western blot或ELISA实验，花费宝贵的样品，以解剖这些信号通路。我们在这里描述了一种新的，高通量，超灵敏的方法，用于同时检测多种磷酸化蛋白。该方法包括一个三明治免疫测定，由多个泛特异性捕获抗体和针对磷酸化和非磷酸化靶蛋白表位的鸡尾酒检测抗体组成。每个检测抗体都有一个独特的DNA标签。实时聚合酶链式反应（Real-time PCR）随后被用来定量所有的DNA标记，从而揭示生物样品中同源（磷-）蛋白的数量。我们发现这种免疫pcr方法比ELISA更敏感，可以在真实生物样品中检测亚皮摩尔水平的内源性蛋白质。在这项拨款申请中，我们建议通过开发一种高灵敏度、多路复用的方法来测量BCR-ABL通路中信号蛋白的磷酸化状态，从而发展和验证这一概念。该平台将允许研究人员从最小（~50 ul）样品体积测量大量的磷酸化蛋白。由于所有步骤都使用微量滴度板和简单的操作，这项技术将允许研究人员以最小的努力和标准设备，破译由众多生物刺激引起的磷酸化模式的变化。

DESCRIPTION (provided by applicant): Site-specific protein phosphorylation is essential for signal transduction in mammalian cells, and perturbation in phosphoprotein levels is a hallmark of cancerous transformation. Unfortunately there are no sensitive methods for detecting multiple phosphorylation events in parallel, so researchers are forced to run multiple Western blot or ELISA experiments, expending precious samples, in order to dissect these signaling pathways. We describe here a novel, high throughput, ultrasensitive method for detecting multiple phosphoproteins simultaneously. The method consists of a sandwich immuno- assay comprised of multiple pan-specific capture antibodies and a cocktail of detection antibodies against both phosphorylated and non-phosphorylated epitopes of the target proteins. Each detection antibody is labeled with a unique DNA tag. Real-time PCR is then used to quantify all of the DNA tags in parallel, thus revealing the quantities of the cognate (phospho-)proteins in the biological samples. We show that this immuno-PCR method is more sensitive than ELISA and can detect endogenous proteins at sub-picomolar levels in real biological samples. In this grant application, we propose to develop and validate this concept by developing a highly sensitive, multiplexed assay to measure the phosphorylation states of signaling proteins in the BCR-ABL pathway. This platform will allow researchers to measure numerous phosphoproteins from minimal (~50 ul) sample volumes. Because all steps use microtiter plates and simple manipulations, this technology will allow researchers, with minimal effort and standard equipment, to decipher changes in phosphorylation patterns resulting from numerous biological stimuli.