Label-Free Microarray Profiling of Phosphoinositide-PDZ Domain Interactions

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DESCRIPTION (provided by applicant): High-throughput microarray technology has become exceedingly promising and important in proteomics research. It allows parallel, large-scale investigation of protein interactions, enabling thousands of compounds to be studied en masse. Protein and antibody microarray technologies are two most propitious technologies for the screening of complex protein samples. However, many limitations of the technology are still unsolved, which have prevented protein microarray technology from reaching its full potential. Proteomics studies are limited by problems such as sample preparation and data analysis, especially in conservation of functionality of capture proteins during immobilization and provision of sensitive detection methods. In recent years, surface plasmon resonance (SPR) imaging has evolved to become a very attractive detection method in microarray analysis. It offers high detection sensitivity without the need of a label, and enables various measurement functions, including real time detection and kinetic analysis to be carried out with simple instrument configuration and operational procedure. In this respect, the technique is unsurpassed by any existing method in microarray technology. Nevertheless, problems in obtaining high quality arrays and non-specific protein interactions have impeded the wide acceptance of SPR imaging method in microarray analysis. Lipid microarray may offer a unique niche for advancing high throughput protein screening technique. Many cellular functions and signaling start with the binding events that take place between lipids and the proteins. For instance, phosphoinositides (PIP) exert their effect as signaling molecules and second messengers by directing protein translocation and the formation of macromolecular signaling complexes at specific subcellular locations. Lipid microarrays will allow researchers to obtain a comparable fingerprint of the proteins from a cell or tissue that bind to lipids, and enable the identification of functionally important lipid-binding proteins. Compared to the matured DNA microarray method and fast improving protein microarrays, lipid microarray technology is still only in its infancy. Much of this can be attributed to the great difficulty in array fabrication. The overall goal of this project is to develop new optical substrates for label-free SPR detection with membrane microarrays, and carry out a high throughput analysis to profile interactions of phosphoinositides with PDZ (postsynaptic density protein, disc large, zonula occludens) domains. Phosphoinositides are essential regulators of nuclear functions and membrane trafficking and are associated with cancers and type II diabetes. Specific aims in this proposal are as follows: 1) Design and fabrication of microarray templates with SiOx- coated SPR chips. 2) Formation and characterization of membrane microarrays. 3) Investigation of lipid-protein interactions with lipid microarray/SPR. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Phosphoinositides (PIPs) are known to be associated to stabilization of adhesion structure, targeting and organization of large signaling complexes, and establishment of cell polarity. Their functions are being studied in the context of cancer and neurobiology. Understanding of the nature of PIP-protein interactions will also facilitate the development of new inhibitors of PIP-metabolizing enzymes. These inhibitors can potentially be used as anticancer drugs. There is a tremendous need in designing improved technology for high-throughput methods in PIP research. The proposed microarray technology will significantly enhance the capability to carry out effective approaches in PIP research and thus improve the throughput in screening. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page