Obtaining optimum sensitivity in detection of low abundant proteins is one of major obstacles in proteomics studies. One solution to this problem is to use the natural fractions present in the cells and indeed nowadays analysis of organelle proteomics constitutes a major avenue in this field. In this context chloroplast is one the most important plant organelles that has been receiving attention for many reasons .In addition to supporting photosynthesis, chloroplasts also synthesize hormones, fatty acids and lipids, amino acids, vitamins (B1, K1, and E), nucleotides, secondary metabolites such as alkaloids and isoprenoids, and are required for nitrogen and sulfur assimilation. Pomegranate ( Punica granatum ) that has been under study in this project is a plant rich of valuable secondary metabolites with significant medical effects such as anticancer, antioxidant and antimicrobial activities. With remark to the potential value of pomegranate in producing these compounds; in the present study pomegranate chloroplast was isolated by mechanical homogenization followed by Percoll gradient. In addition, tobacco chloroplast was also similarly purified to serve as a model system for isolation and examination of the chloroplasts. Both isolated chloroplasts were shown to be free of other organelles by Western analysis utilizing antibodies specific to mitochondrion, cytosol and plasma membrane. Comparison of chloroplast isolation in pomegranate and tobacco indicated the presence of interfering phenolic compounds in pomegranate extract that were subsequently dealt with by addition of 5% PVP to the chloroplast isolation buffer. It was also found that pomegranate leaves might yield half the chloroplast amount compared to that from the tobacco leaves. Furthermore preliminary analysis indicated that the protein profile of pomegranate and tobacco were rather different as judged by SDS-PAGE. Following pomegranate chloroplast isolation and protein extraction, chloroplast proteome was resolved using 2D PAGE, from which 4 protein spots were isolated and analyzed by de novo sequencing. These proteins were identified as a ~53-kDa ATP synthase beta subunit, a probable ~70-kDa malate dehydrogenase and a probable ~55-kDa Beta-amylase. Fourth protein spot could not be identified. Literature review shows that this report constitutes the first isolation and proteomics analysis of pomegranate chloroplast.
