Effects of Protease Inhibitors on the Proteins in Two Dimensional Electrophoresis Maps of Hoya carnosa Mitochondria

The analysis and characterizations of complex protein mixtures are the central aims of proteomics. The core technology of proteomics is 2DE which simultaneously separates and displays hundreds to thousands of proteins. In the 2DE technique, the sample preparation step with high protection against proteolysis is one of the necessary steps for high quality resolution of proteins in 2DE maps. The power of 2DE as a biochemical separation technique has virtually been recognized since its introduction. Its application, however, has significantly increased in the last few years. Due to the great diversity of protein sample types and origins, the optimal procedure for sample preparation in 2DE must be determined empirically for new sample source (Berkelman et al., 1998). In deed, some methodologies have been described to extract mitochondrial protein for 2DE; however, the sample preparations in these methods are varied depending on sample types and species. While many methodologies had been described to alleviate these problems, the definition of the optimal conditions for sample preparation from every new cell source is still somewhat of an art In this study, we firstly probed the protein expression on the two dimensional electrophoresis (2DE) maps of Hoya carnosa mitochondria during CAM phase III. Then, we investigated the effects of some protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin (Leu), and monoiodoacetate (MIA) on 2DE maps. Mitochondrial proteins were extracted in the lysis buffer with and without one of the protease inhibitors. The mitochondrial proteins from 2DE maps were surveyed by using Image Master 2DE software (Amersham Pharmacia Biotech, USA). The results indicated that the protease inhibitors clearly affected on the number proteins in the mitochondrial 2DE maps.