Comparative proteomics: characterization of a two-dimensional gel electrophoresis system to study the effect of aging...

To study the effect of aging and anti-aging strategies on mitochondria, we have characterized a two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system to analyze the profile of mitochondrial proteins. We have optimized the separation of proteins by 2-D PAGE and established the linearity and reproducibility of the system with mitochondria isolated from skeletal muscle of mice. Using total mitochondria protein ranging from 10 to 200 microg, we found that 74% of the proteins resolved by 2-D PAGE had coefficient of determination (R2) values greater than 0.8, showing a linear increase in fluorescence with increasing protein concentration. The coefficient of variation (CV) was less than 50% for at least 93% of the 424 spots analyzed for both gel-to-gel variance and animal-to-animal variance. Using mitochondrial protein fractions prepared from skeletal muscle of 18-month-old mice, we show that 10 animals will be sufficient to detect a 100% difference in the 97% (i.e. 505) of the proteins resolved by 2-D PAGE. Thus, 2-D PAGE provides a sensitive and reliable technique for analysis of protein expression in mitochondria.