title: Mining the soluble chloroplast proteome by affinity chromatography.
abstract: Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.
authors.affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
authors.forename: Roman G
authors.initials: RG
authors.lastname: Bayer
authors.forename: Simon
authors.initials: S
authors.lastname: Stael
authors.forename: Edina
authors.initials: E
authors.lastname: Csaszar
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity / Q000379:methods*; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism; D030541:Databases, Genetic; D020224:Expressed Sequence Tags / Q000737:chemistry; D015870:Gene Expression; D018613:Microscopy, Confocal; D018532:Pisum sativum / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010736:Phosphoglycerate Mutase / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D012995:Solubility; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
chemical_list: D010940:Plant Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D057056:Cysteine Proteases; D012273:Ribulose-Bisphosphate Carboxylase; D010736:Phosphoglycerate Mutase