title: Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses.
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 (http://www.promexdb.org/home.shtml). 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-CO2-inducible mitochondrial CAH isoform. This indicates its role as one of the strongest CO2-responsive proteins in the crosstalk of air-adapted mixotrophic chloroplast and mitochondrial metabolism in Chlamydomonas reinhardtii.
authors.affiliation: Dept. of Molecular Systems Biology, University of Vienna, Vienna, Austria. stefanie.wienkoop@univie.ac.at
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wienkoop
authors.forename: Julia
authors.initials: J
authors.lastname: Weiss
authors.forename: Patrick
authors.initials: P
authors.lastname: May
authors.forename: Stefan
authors.initials: S
authors.lastname: Kempa
authors.forename: Susann
authors.initials: S
authors.lastname: Irgang
authors.forename: Luis
authors.initials: L
authors.lastname: Recuenco-Munoz
authors.forename: Matthias
authors.initials: M
authors.lastname: Pietzke
authors.forename: Thorsten
authors.initials: T
authors.lastname: Schwemmer
authors.forename: Jens
authors.initials: J
authors.lastname: Rupprecht
authors.forename: Volker
authors.initials: V
authors.lastname: Egelhofer
authors.forename: Wolfram
authors.initials: W
authors.lastname: Weckwerth
mesh_terms: D000085:Acetates / Q000378:metabolism; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D030562:Databases, Protein; D023281:Genomics / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D055432:Metabolomics / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism; D045331:Photosystem I Protein Complex / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000085:Acetates; D020418:Algal Proteins; D002247:Carbon Isotopes; D045331:Photosystem I Protein Complex; D045332:Photosystem II Protein Complex; D020543:Proteome; D002256:Carbonic Anhydrases