Targeted Proteomics for Chlamydomonas reinhardtii combines mass western, subcellular protein fractionation, metabolomics and metabolic flux analysis
- Author(s)
- Stefanie Wienkoop, Julia Weiß, Patrick May, Stefan Kempa, Susann Irgang, Luis Recuenco-Munoz, Matthias Pietzke, Thorsten Schwemmer, Jens Rupprecht, Volker Egelhofer, Wolfram Weckwerth
- 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.
- Organisation(s)
- External organisation(s)
- Max-Planck-Institut für Molekulare Pflanzenphysiologie
- Journal
- Molecular BioSystems
- Volume
- 6
- Pages
- 1018-1031
- No. of pages
- 14
- ISSN
- 1742-206X
- DOI
- https://doi.org/10.1039/b920913a
- Publication date
- 2010
- Peer reviewed
- Yes
- Austrian Fields of Science 2012
- 106037 Proteomics, 106023 Molecular biology
- Portal url
- https://ucris.univie.ac.at/portal/en/publications/targeted-proteomics-for-chlamydomonas-reinhardtii-combines-mass-western-subcellular-protein-fractionation-metabolomics-and-metabolic-flux-analysis(5fdb57b3-2f32-481c-a668-d7bc1df8735f).html