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.

Influence of CO2concentration on carbon concentrating mechanisms in cyanobacteria and green algae: a proteomic approach

“…Among algae in general, the only proteome to be studied is that of Chlmaydomonas reinhardtii, with even with sub-cellular proteome investigations (Stauber and Hippler 2004, Atteia et al 2009, Wienkoop et al 2010. Recently, C. reinhardtii proteome, transcriptome and metabolome have been studied under varying CO 2 concentration.…”

Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change

“…Regulation here means the changes to the functioning of the pre-existing proteome (changes in post-translational modification and in ligand concentration) over times of seconds to minutes of a change in conditions: there is not enough time for changes to the proteome, related to changes in transcription and translation. Acclimation is defined as changes in the use of the existing genome by changes in transcription and translation, and hence in the proteome and metabolome (Raven 2010(Raven , 2011Renberg et al 2010;Wienkoop et al 2010), in response to changes in the environment; it occurs over time intervals of an hour and longer, and occurs in parallel with, and may modulate, regulation. Adaptation is taken to mean evolutionary changes to the genome in response to changed environmental conditions, with the possibility of more extreme changes to the proteome and metabolome than is the case for acclimation.…”

“…Inorganic carbon is supplied from weathering of rocks and input of CO 2 derived from breakdown of organic carbon fixed in the catchment. The catchment can also supply large amounts of terrestrially-produced dissolved organic carbon (DOC), often with high short-wavelength absorbance, that can ameliorate harmful effects of UVB (Williamson et al 1996) but also limit productivity by absorbing PAR (Karlsson et al 2009). Microbial and photochemical degradation of DOC within a lake is one of the causes of the generally elevated concentrations of CO 2 in many lakes (Tranvik et al 2009).…”

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

“…When integrated with enzyme data, metabolite profiling can reveal the activity in parts of the metabolic network (27). However, only a few investigations on metabolomic and enzymatic data integration have been conducted in microalgae (23,28,29), and a systems-based study of the carbon-nitrogen metabolism in response to nitrogen starvation under HL in H. pluvialis is still lacking.…”