This proposal is to develop a combined preparative-analytical two-dimensional zonal ultracentrifuge system, based on early prototypes developed at Oak Ridge, to both measure accurately the sedimentation rates (s) and isopycnic banding densities (p) of cells, subcellular organelles and infectious agents, and to perform high resolution preparative separations based on these parameters. The purified fractions will be anaylyzed by high resolution two-dimensional electrophoresis with the objective of assigning to each protein that can be detected a specific subcellular location. Initial demonstration and feasibility studies will be concerned with separating and analyzing different classes of liver and brain nuclei. LSB is currently preparing standard reference maps of human and rodent proteins by organ, is identifying proteins by composition or sequence, and is mapping the effects of drugs, hormones, and toxic agents of gene expression by 2DE. We now require methods for producing subcellular fractions (nuclei, mitochondria, peroxisomes, lysosomes, etc.) reproducibly, and for further subfractionating these into nuclear membranes, nucleoli, nuclear pore complexes, inner and outer mitochondrial membranes, etc., so that the location of each protein gene product can be determined. The 2D centrifuge systems proposes will also be useful for the detection and isolation of infectious agents from tissues and body fluids.
