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. ? ? ?