HIGH-THROUGHPUT PROTEOMIC ANALYSIS OF SIGNALING PATHWAYS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Emerging methodologies in mass spectrometry derive their utility through application to persistent, difficult problems in pharmacology and cellular biology. The ability of the mass spectrometer to assign sites of phosphorylation in complex mixtures of peptides represents an unique opportunity to greatly accelerate the elucidation of signaling pathways. Here we propose the development of a phosphoproteomic technology platform capable of the automated analysis of sites of phosphorylation derived from whole cell lysates. We will exploit this platform to discern the architecture of signaling pathways relevant to mast cell and hepatocellular carcinoma (HCC) signaling. Identification of novel signaling pathway members will provide targets for rational development of drugs that selectively inhibit the pathways. The long term goals of the proposed new studies are: (1) to exploit a state-of-the-art mass spectrometry technology platform to determine all of the proteins, their sites of phosphorylation, and the temporal and spatial pattern of phosphorylation involved in cell signaling; (2) to increase the sensitivity of detection of extremely low abundance signaling molecules from complex cell lysates; (3) to develop bioinformatic tools for the visualization, organization, and storage of large phosphoproteomic data sets. Our specific aims are (1) to deploy a technology platform to determine the signaling pathway-related phosphorylation events and illustrate its viability with mast cell activation; (2) to identify the key phosphorylation events important in HCC cell proliferation and migration; (3) to develop and thoroughly test bioinformatic and analytical improvements to our phosphoproteomic platform.