Affinity Interactions in Capillary Separations

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

In this project supported by the Analytical and Surface Chemistry Program, Professor Robert Kennedy and co-workers at the University of Florida will develop novel analytical methods that allow proteins to be detected and quantified based on their affinity for natural binding partners. In the methods, fluorescently-labeled ligands for classes of proteins will be added to samples and the resulting complexes will be separated by capillary electrophoresis and detected with laser-induced fluorescence. Binding systems that will be investigated include labeled GTP to detect G-proteins, NADH to detect dehydrogenases, labeled phosphopeptides to detect proteins containing src homology 2 domains, and src homology 2 domains to detect phosphorylated proteins. The method is expected to provide high sensitivity (attomole detection limits are expected), high speed (analysis on the second to minute time scale are possible) and facile automation. The methods are expected to be useful for tracking the expression level of proteins or for discovery of novel proteins and binding partners of proteins. As signal transduction in biological systems relies extensively on molecular recognition, these affinity methods are expected to be especially useful in experiments aimed at uncovering the regulation of receptor-mediated changes in cells. The methods will be tested on pancreatic beta-cells where they may ultimately be useful in developing a better understanding of insulin secretion The sequencing of the genome has provided a blueprint for life. The blueprint is actually the code for all of the proteins that are made in a cell. Cells are not readily defined however even by all of the proteins that they make. What are the functions of the proteins? How do they interact with each other? How are the interactions and functions controlled? A short answer to these questions is that proteins are controlled by their affinity i.e., ability to bind selected targets or partners within the cell. In this project, a method is sought that allows proteins to be rapidly and sensitively detected based on their affinity for a particular molecule. Importantly, the method will allow all the proteins with a given affinity to be separated and detected. The new method will be at least 1000 times as sensitive as existing methods and can be performed in seconds or minutes rather than hours. This new method will allow new proteins or protein mixtures to be rapidly screened for certain binding properties or functions. It will also allow classes of known proteins to be rapidly assayed. The speed and sensitivity of the assays will enable applications in diverse fields such as drug discovery, signal transduction (i.e., understanding intracellular chemical communication), and biotechnology.