Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics

Posidonia oceanica is a marine angiosperm, or seagrasses, adapted to grow to the underwater lifein shallow waters as at 40, 50 m depth in very clear waters. This pose many questions on how theirphotosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolisms of the chloroplast are the bases of adaptive capacity.Obtaining seagrass chloroplast proteome is central to increase the knowledge about the fundamental biology of marine plastids. We modified existing protocols optimized in terrestrial plants to extract the organelles from the minimal tissue amount as possible; we obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant; chloroplasts were purified from the other cell components by isopynic separations. Organelle proteins were extracted fromhigh purified chloroplasts and resolved by 1DE SDS-PAGE. Proteins sequencing was made by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against nrNCBI green plant databases and a local customized dataset. The assignments of chloroplast proteins in the sub-compartment envelope, stroma and thylakoids were validated by peptide searching against the AT_CHLORO database. On this basis we evaluated the yield of protein extraction and identification performance in each compartment.