Meeting Abstract

P2.120  Jan. 5  Two-dimensional gel electrophoresis of heat-denatured and ubiquitylated proteins in Mytilus californianus LABARGE, J.*; TOMANEK, L.; California Polytechnic State Univ., San Luis Obispo jeremylabarge@gmail.com

The Ubiquitin Proteasome Pathway (UPP) is the primary metabolic pathway used to degrade denatured proteins. The UPP operates by binding the small protein ubiquitin to denatured proteins by way of an enzyme cascade. Ubiquitin-conjugated proteins are transported to a proteolytic complex, called the 26S proteasome, where degradation takes place. By inhibiting the proteasome we can enrich proteins that are denatured due to thermal stress and that are subsequently conjugated with ubiquitin. As a first step we exposed gill tissue of the intertidal mussel Mytilus californianus to heat shock-inducing temperatures and subsequently separated proteins, first, by isoelectric focusing, and, second, by gel electrophoresis (two-dimensional gel electrophoresis). We detected over 700 different protein spots in the range of pH 4-7. Using western blotting and affinity chromatography we are currently identifying the protein spots that are tagged with a single or multiple ubiquitin molecule(s) due to denaturation following heat stress. Ubiquitylated proteins will be excised, digested with trypsin and analyzed by peptide mass fingerprinting and de novo protein sequencing with a MALDI-TOF-TOF mass spectrometer, in order to identify the proteins that have been denatured by thermal stress. With our findings we hope to find a set of heat sensitive proteins that are critical to setting the thermal limits of mussel species of the genus Mytilus.