Abstract
The chloroplast is an intracellular factory where major substrates such as sugars, amino acids, lipids, terpenoids, carotenoids, and porphyrins all necessary for cellular functions are produced, in addition to photosynthetic activity with utilizing the solar energy.
We can isolate intact chloroplasts as free as possible from the other intracellular components by repeating Percoll gradient centrifugation, minimizing the contamination with catalase as a marker enzyme for peroxisomes or fumarase for mitochondria less than 1.0%. An anhydrotrypsin column was employed for enriching C-terminal peptides from the mixture of trypsin-digests of the chloroplast proteins. The collected peptides were further subjected to two-dimensional HPLC/MS/MS [MudPIT (Multidimensional Protein Identification Technology)], resulting in identification of approx. 4,600 species in the criterion of DeltCN higher than 0.1, and over 1.200 species with Xcorr > 2.0 and ion > 40%.
