Purification and proteomic analysis of chloroplasts and their sub-organellar compartments.
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.
Salvi
Daniel
D
Laboratoire de Physiologie Cellulaire Végétale, Grenoble, France.
Rolland
Norbert
N
Joyard
Jacques
J
Ferro
Myriam
M
D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D006056:Golgi Apparatus / Q000737:chemistry* / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008099:Liver / Q000648:ultrastructure; D008322:Mammals; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome; D051381:Rats; D014357:Trypsin
D007202:Indicators and Reagents; D011506:Proteins; D020543:Proteome; D014357:Trypsin