This proposal is designed to develop zwitterion mixtures that have the same properties from batch to batch. It is well known among investigators using 2D electrophoresis that different lots of carrier ampholytes have unpredictable variations in quality and that results vary depending on the source and lot of ampholyte. This is largely due to the variations that occur in the building blocks used for synthesis of the ampholytes. Recent data show that simple hydration of the polyamines used to synthesize ampholytes can yield >95% pure linear forms of the molecules while eliminating branched and ring structures from the preparations. Thus, the availability of chemically pure starting material should allow synthesis of ampholyte mixtures that do not vary from lot to lot. Protocols for synthesis of carrier ampholytes will be developed that are based on preliminary data to create highly complex ampholyte mixtures covering wide pH intervals (pH 3.5-10) and narrow range intervals covering as little as one pH unit. Purified linear forms of polyamines will first be cross-linked with divalent ester compounds to create structurally complex polyamine mixtures of different sizes and isomers. These mixtures will then be reacted with acrylic acid to create highly heterogeneous polyamino-polycarboxylic acid mixtures of molecules covering the whole pH spectrum from pH 3.5 to pH 10. Narrow range ampholytes will be synthesized directly by first altering the polyamine molecules by reaction with acrylamide or acrylonitrile followed by reaction with optimal ratios of acrylic acid. Maximum heterogeneity of mixtures will be monitored using patented technology for visualizing the distribution of ampholytes in gels by separating ampholytes on immobilized pH gradients and precipitating them in place using picric acid. Patterns of ampholyte distribution will be analyzed by image analysis densitometry to assure smooth uninterrupted distribution across the pH gradient. The innovative nature of this project is that successful completion of the work will result in the availability of carrier ampholytes for 2D electrophoresis and gel isoelectric focusing that have no variation from lot to lot. This would have a major impact on the fields of Functional Genomics and Proteomics by allowing 2D electrophoresis techniques to be standardized in far more accurate and reproducible ways than is possible with the commercial ampholytes that are currently available.
