title: Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.
abstract: Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.
authors: Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany. Electronic address: pklundquist@noble.org.
authors: Peter K
authors: PK
authors: Lundquist
authors: Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors: Otho
authors: O
authors: Mantegazza
authors: Molecular Proteomics Laboratory, BMFZ, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors: Anja
authors: A
authors: Stefanski
authors: Molecular Proteomics Laboratory, BMFZ, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors: Kai
authors: K
authors: Stühler
authors: Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, 40225 Düsseldorf, Germany.
authors: Andreas P M
authors: APM
authors: Weber
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D050356:Lipid Metabolism; D009154:Mutation; D018515:Plant Leaves / Q000737:chemistry; D040901:Proteomics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D020524:Thylakoids / Q000737:chemistry
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins
keywords: RETICULATA; blue native-PAGE; chloroplast; comparative proteomics; oligomeric state; protein complex