Live cell affinity receptor chromatography (LARC)

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Cell surface receptors are a key group of proteins targeted by drugs for the treatment of disease. Cell surface receptors may bind proteins or other target molecules that carry messages to the cell. There has been a concerted, but largely unsuccessful, effort to find a way to directly isolate and analyze the proteins in receptor complexes since the middle of the last century. I propose to create a method to directly capture a receptor complex from the surface of live cells by fixing their binding target onto microscopic beads. I will use a protein from blood to capture its receptor complex from the surface of living white blood cells. Human white blood cells have receptors that bind to germs to engulf and destroy them. Antibodies for bacterial recognition may be attached to microscopic beads. The beads will be used to capture the receptor complex for engulfing germs from the surface of the white blood cells. The microbeads carrying the receptor beads will be collected and the receptor proteins captured will be identified by mass spectrometry. Different types of salts and detergents as well as enzymes will be used to remove common cellular material such as DNA, RNA or ribosomes that tend to stick to beads. The optimal type of bead, size of bead, chemical method of attachment to the bead, and time for collecting the beads will be determined. The best methods for extracting the receptor proteins and to fractionate them using chromatography prior to identification will be determined. The proteins can be identified by breaking them into pieces with enzymes, and separating them followed by mass spectrometry and computer searching using classical mathematics. Proteins identified from extracted receptor complexes will be confirmed with a laser microscope by independent methods such as the use of specific antibodies and green fluorescence tagging. The method will elucidate the proteins required by the receptor complex for engulfing germs. More importantly, our success would mean that Canada will have a general method for isolating receptor complexes directly from the surface of live cells. This will permit the identification and validation of new and specific drug targets for the treatment of many diseases.