Ultrasensitive pathway phosphoproteomics

描述(申请人提供)：特定部位的蛋白质磷酸化对于哺乳动物细胞的信号转导是必不可少的，而磷蛋白水平的扰动是癌症转化的标志。不幸的是，目前还没有灵敏的方法来同时检测多个磷酸化事件，因此研究人员被迫进行多个Western印迹或ELISA实验，花费宝贵的样本来剖析这些信号通路。我们描述了一种新的、高通量、超灵敏的同时检测多个磷酸蛋白的方法。该方法由多个PAN特异性捕获抗体和针对目标蛋白的磷酸化和非磷酸化表位的检测抗体的鸡尾酒组成的夹心免疫分析。每个检测抗体都标有唯一的DNA标签。然后用实时荧光聚合酶链式反应对所有DNA标签进行并行定量，从而揭示生物样品中同源(磷酸)蛋白的数量。我们发现，这种免疫-聚合酶链式反应方法比酶联免疫吸附试验更灵敏，可以在真实生物样品中检测到亚皮摩尔水平的内源蛋白。在这项资助申请中，我们建议开发和验证这一概念，通过开发一种高度灵敏的、多重的方法来测量BCR-ABL途径中信号蛋白的磷酸化状态。该平台将允许研究人员从最小(~50 ul)样本体积中测量大量磷蛋白。由于所有步骤都使用微滴定板和简单的操作，这项技术将允许研究人员以最少的努力和标准设备，破译由于大量生物刺激而导致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.