• Establishment of proteome dynamics of rat mitochondrial proteins

      Shekar, Kadambari Chandra; Stanley, William C., Ph.D. (2012)
      Proteomics has provided a major advancement in research due to rapid identification and quantification of a relatively large number of proteins. However, most of the current methods measure only the static protein levels. Proteome dynamics is the method of studying protein synthesis and/or degradation rates using isotopic tracers and advance mass spectrometry. In our study, we used heavy water (2H2O) as a stable isotopic tracer to measure the synthesis rates of various cardiac and liver mitochondrial proteins. Adult male Sprague-Dawley rats were administered with 5% heavy water in drinking water for pre-selected time points that include 0,3,10,20,40 and 60 days. Food and water were given ad libitum during this period. The rats were then euthanized, their heart and liver tissues harvested. Liver mitochondria and the two subpopulations of cardiac mitochondria including sub-sarcolemmal mitochondria (SSM) and inter-fibrillar mitochondria (IFM) were isolated by homogenization and centrifugation. Proteins from these subpopulations were separated by gradient gel electrophoresis, selected gel bands were then excised, tryptically digested and analyzed using a mass spectrometer for identification and determination of the protein synthesis rates. We were able to measure the synthesis rates of various nuclear DNA encoded and mitochondrial DNA encoded cardiac proteins with half-lives ranging from 17-45 days. On the other hand, liver proteins had a significantly faster turnover, with half-lives around 4-6 days. Future studies include comparing the protein synthesis rates of healthy rats and rats with heart failure in order to identify potential biomarkers of heart failure.