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Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses

Item Type:Article
Title:Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses
Creators Name:Wienkoop, S. and Weiss, J. and May, P. and Kempa, S. and Irgang, S. and Recuenco-Munoz, L. and Pietzke, M. and Schwemmer, T. and Rupprecht, J. and Egelhofer, V. and Weckwerth, W.
Abstract:In the era of fast genome sequencing a critical goal is to develop genome-wide quantitative molecular approaches. Here, we present a metaproteogenomic strategy to integrate proteomics and metabolomics data for systems level analysis in the recently sequenced unicellular green algae Chlamydomonas reinhardtii. To achieve a representative proteome coverage we analysed different growth conditions with protein prefractionation and shotgun proteomics. For protein identification, different genome annotations as well as new gene model predictions with stringent peptide filter criteria were used. An overlapping proteome coverage of 25%, consistent for all databases, was determined. The data are stored in a public mass spectral reference database ProMEX (). A set of proteotypic peptides comprising Calvin cycle, photosynthetic apparatus, starch synthesis, glycolysis, TCA cycle, carbon concentrating mechanisms (CCM) and other pathways was selected from this database for targeted proteomics (Mass Western). Rapid subcellular fractionation in combination with targeted proteomics allowed for measuring subcellular protein concentrations in attomole per 1000 cells. From the same samples metabolite concentrations and metabolic fluxes by stable isotope incorporation were analyzed. Differences were found in the growth-dependent crosstalk of chloroplastidic and mitochondrial metabolism. A Mass Western survey of all detectable carbonic anhydrases partially involved in carbon-concentrating mechanism (CCM) revealed highest internal cell concentrations for a specific low-CO(2)-inducible mitochondrial CAH isoform. This indicates its role as one of the strongest CO(2)-responsive proteins in the crosstalk of air-adapted mixotrophic chloroplast and mitochondrial metabolism in Chlamydomonas reinhardtii.
Keywords:Acetates, Algal Proteins, Carbon Isotopes, Carbonic Anhydrases, Chlamydomonas reinhardtii, Chloroplasts, High Pressure Liquid Chromatography, Cytosol, Protein Databases, Genomics, Mass Spectrometry, Metabolomics, Mitochondria, Photosystem I Protein Complex, Photosystem II Protein Complex, Proteome, Proteomics, Subcellular Fractions
Source:Molecular BioSystems
Publisher:Royal Society of Chemistry
Page Range:1018-1031
Date:18 June 2010
Official Publication:https://doi.org/10.1039/b920913a
PubMed:View item in PubMed

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