Sub-cellular proteomics has proven to be a powerful approach to link the information contained in sequenced genomes from eukaryotic cells to the functional knowledge provided by studies of cell compartments. Chloroplasts are plant-specific organelles and are the site of photosynthesis and also of many other essential metabolic pathways, like syntheses of amino acids, vitamins, and pigments. They contain several sub-organellar compartments: the envelope (the two-membrane system surrounding the organelle), the stroma (the internal soluble phase), and the thylakoid membranes (the internal membrane system). There is a link between these compartments and the functions of their constitutive proteins. One way to bring into view the sub-proteomes of the chloroplast is to develop proteomic analyses based (1) on the use of highly purified sub-fractions of the chloroplast and (2) on mass spectrometry (MS)-based analyses for protein identification. To illustrate such strategies, this chapter describes the methods for purification of chloroplasts from Arabidopsis leaves and for the specific recovery of highly pure sub-organellar fractions of envelope, stroma, and thylakoids. Subsequently, methods are described to analyze by MS the proteins recovered from these fractions.
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