The goal of this project is to greatly enhance the sensitivity, information content, and throughput of proteomics by developing a family of optimized designer fluorescent dyes and coupling them with multiplex 2D separation and detection methods to identify proteins associated with cellular functions. Proteomic methodologies, as commonly practiced using 2D gels, are able to detect proteins down to about 300-1,000 copies per cell, a level insufficient to monitor many important cellular control proteins that are present in low copy numbers. 2D gels reveal global patterns of protein expression, but suffer from gel-to-gel reproducibility problems that limit the ability to detect changes in protein expression and post-translational modification, which are associated with biological mechanisms in health and disease. ? ? The proposed new fluorescent dyes are designed to: (I) increase the solubility of dye-labeled proteins for reduction of gel artifacts and increased loading capacity, (II) enhance sensitivity ca. 300x to allow detection of proteins in the range of 1-3 copies per cell, and (III) allow multicolor, multiplex separation and detection of proteins produced by cells under different physiological conditions simultaneously on the same gel, to overcome gel-to-gel reproducibility problems. This multiplex separation and detection technique significantly reduces the need for brute force characterization of all the proteins in cells, by revealing proteins that are up or down regulated under particular physiological or disease conditions or stimuli of interest. ? ? The new dyes are designed to label abundant surface lysine residues on proteins for maximum sensitivity and variants of the dyes have been designed that can be removed efficiently under mild conditions to allow trypsin digestion for mass analysis. Alternatively, for proteins migrating in crowded regions on 2D gels, the dyes can be left on the proteins during a 3rd dimension HPLC separation with sensitive fluorescence detection and then removed when proteolysis is needed for mass spectral analysis. ? ? The new methods promise to provide major increases in understanding of fundamental elements of virtually every biomedical phenomena, including (but not limited to) developmental mechanisms, signaling networks and responses to stress and infection--and are likely to lead to new commercial products.
| Barry, Richard C; Young, Mark J; Stedman, Kenneth M et al. (2006) Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2. Electrophoresis 27:2970-83 |
| Barry, Richard C; Alsaker, Britt L; Robison-Cox, James F et al. (2003) Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis. Electrophoresis 24:3390-404 |
