High-Throughput Measurement of Murine Mitochondrial Protein Turnover by Metabolic Heavy Water (2H2O)Labeling
소속 :
연사 : 김태영 박사님
일시 : 2012-09-27 10:30 ~
장소 : 503동 404호
일 시 : 2012년 9월 27일, 10:30 AM
??소 : 503동 404호
-Abstract-
Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage exacerbates under inadequate protein quality control, and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of its continuous protein synthesis and degradation, i.e., protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (2H2O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with 2H2O at a minimal level (<5% body water) without physiological impacts. Murine mitochondrial proteins were analyzed at each of the 13 time points between 0 and 90 d of labeling. A novel multi-parameter fitting approach computationally determines the normalized peak areas of peptide mass isotopomers at initial and steady-state time points, and permits protein half-life to be determined without plateau-level 2H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria, with median turnover rates of 0.0402 and 0.163 d-1, respectively, corresponding to median half-lives of 17.2 and 4.26 d. Mitochondria in the heart and the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable inter-protein differences in turnover rates in both organs, with half-lives spanning from hours to months (~60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.
??소 : 503동 404호
-Abstract-
Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage exacerbates under inadequate protein quality control, and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of its continuous protein synthesis and degradation, i.e., protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (2H2O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with 2H2O at a minimal level (<5% body water) without physiological impacts. Murine mitochondrial proteins were analyzed at each of the 13 time points between 0 and 90 d of labeling. A novel multi-parameter fitting approach computationally determines the normalized peak areas of peptide mass isotopomers at initial and steady-state time points, and permits protein half-life to be determined without plateau-level 2H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria, with median turnover rates of 0.0402 and 0.163 d-1, respectively, corresponding to median half-lives of 17.2 and 4.26 d. Mitochondria in the heart and the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable inter-protein differences in turnover rates in both organs, with half-lives spanning from hours to months (~60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.
