The phosphorylation, or activation state of kinase-driven signal networks embodies information concerning disease pathogenesis and the ongoing state of kinase associated therapeutic targets. Profiling the tumor phosphoproteome using human tumor biopsy specimens is a crucial component of the perceived upcoming revolution of individualized cancer therapy. A critical unmet need addressed by this application is tissue phosphoprotein stability data, standardized protocols, and novel technologies which can be used in the real world clinical setting (e.g. operating room, outpatient clinic biopsy, radiological suite needle aspiration) for seamless collection, immediate preservation, and real time stability monitoring of phosphoproteins. Our multidisciplinary team has previously developed Laser Capture Microdissection (LCM) and reverse phase protein microarray (RPA) technologies to conduct phosphoproteomic analysis of the tissue microenvironment. We will standardize the RPA technology to simultaneously measure at least 100 known phosphoprotein endpoints with high precision and sensitivity in a human core needle biopsy or fine needle aspirate. This technology will be used to collect previously unknown quantitative information about the tissue half-life of phosphoproteins representing a wide range of signaling pathways, cellular compartments and phosphorylated residues over time zero to 48 hours. These data will become the basis to identify a subset of highly representative and most-labile endogenous phosphoproteins which can be employed as novel surrogate quantitative markers of tissue preservation and phosphoprotein stability. We will employ this stability data and novel surrogate endpoints technology to propose definitive quantitative guidelines for tissue perishability limits. We will create exogenous phosphoprotein sentinel nanoparticles to record the processing history of the specimen for ongoing Quality Assurance. The RPA, surrogate markers, and sentinel technology will be used to rank candidate preservative solutions that stabilize kinases, phosphatases, and phosphoproteins for 24-48 hr at room temperature. The desired outcome will be a complete standardized technology kit which can be employed for routine clinical collection, shipping, early warning of perished tissue, and real time monitoring of tissue phosphoproteins for molecular profiling. ? ? ?
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