RRC ID 12332
Author Bayer RG, Stael S, Csaszar E, Teige M.
Title Mining the soluble chloroplast proteome by affinity chromatography.
Journal Proteomics
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.
Volume 11(7)
Pages 1287-99
Published 2011-4
DOI 10.1002/pmic.201000495
PMID 21365755
PMC PMC3531887
MeSH Adenosine Triphosphate / metabolism Arabidopsis / chemistry Chloroplasts / chemistry Chloroplasts / genetics* Chloroplasts / metabolism Chloroplasts / ultrastructure Chromatography, Affinity / methods* Cysteine Proteases / genetics Cysteine Proteases / metabolism Databases, Genetic Expressed Sequence Tags / chemistry Gene Expression Microscopy, Confocal Peas / chemistry Peas / genetics* Peas / metabolism Phosphoglycerate Mutase / genetics Phosphoglycerate Mutase / metabolism Plant Proteins / analysis Plant Proteins / genetics* Plant Proteins / metabolism Protein Denaturation Protein Kinases / genetics Protein Kinases / metabolism Proteome / analysis* Recombinant Fusion Proteins / genetics* Recombinant Fusion Proteins / metabolism Ribulose-Bisphosphate Carboxylase / metabolism Solubility Tobacco / genetics Tobacco / metabolism Tobacco / ultrastructure Transfection
IF 3.532
Times Cited 24
WOS Category BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Arabidopsis / Cultured plant cells, genes pda13664