Functional analysis of proteins and protein species using shotgun proteomics and linear mathematics.

Covalent post-translational modification of proteins is the primary modulator of protein function in the cell. It greatly expands the functional potential of the proteome compared to the genome. In the past few years shotgun proteomics-based research, where the proteome is digested into peptides prior to mass spectrometric analysis has been prolific in this area. It has determined the kinetics of tens of thousands of sites of covalent modification on an equally large number of proteins under various biological conditions and uncovered a transiently active regulatory network that extends into diverse branches of cellular physiology. In this review, we discuss this work in light of the concept of protein speciation, which emphasizes the entire post-translationally modified molecule and its interactions and not just the modification site as the functional entity. Sometimes, particularly when considering complex multisite modification, all of the modified molecular species involved in the investigated condition, the protein species must be completely resolved for full understanding. We present a mathematical technique that delivers a good approximation for shotgun proteomics data.

Metabolomics- and proteomics-assisted genome annotation and analysis of the draft metabolic network of Chlamydomonas reinhardtii.

We present an integrated analysis of the molecular repertoire of Chlamydomonas reinhardtii under reference conditions. Bioinformatics annotation methods combined with GCxGC/MS-based metabolomics and LC/MS-based shotgun proteomics profiling technologies have been applied to characterize abundant proteins and metabolites, resulting in the detection of 1069 proteins and 159 metabolites. Of the measured proteins, 204 currently do not have EST sequence support; thus a significant portion of the proteomics-detected proteins provide evidence for the validity of in silico gene models. Furthermore, the generated peptide data lend support to the validity of a number of proteins currently in the proposed model stage. By integrating genomic annotation information with experimentally identified metabolites and proteins, we constructed a draft metabolic network for Chlamydomonas. Computational metabolic modeling allowed an identification of missing enzymatic links. Some experimentally detected metabolites are not producible by the currently known and annotated enzyme set, thus suggesting entry points for further targeted gene discovery or biochemical pathway research. All data sets are made available as supplementary material as well as web-accessible databases and within the functional context via the Chlamydomonas-adapted MapMan annotation platform. Information of identified peptides is also available directly via the JGI-Chlamydomonas genomic resource database (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html).

Targeted quantitative analysis of a diurnal RuBisCO subunit expression and translation profile in Chlamydomonas reinhardtii introducing a novel Mass Western approach.

RuBisCO catalyzes the rate-limiting step of CO2 fixation in photosynthesis. Hypothetical mechanisms for the regulation of rbcL and rbcS gene expression assume that both large (LSU) and small (SSU) RuBisCO subunit proteins (RSUs) are present in equimolar amounts to fit the 1:1 subunit stoichiometry of the holoenzyme. However, the actual quantities of the RSUs have never been determined in any photosynthetic organism. In this study the absolute amount of rbc transcripts and RSUs was quantified in Chlamydomonas reinhardtii grown during a diurnal light/dark cycle. A novel approach utilizing more reliable protein stoichiometry quantification is introduced. The rbcL:rbcS transcript and protein ratios were both 5:1 on average during the diurnal time course, indicating that SSU is the limiting factor for the assembly of the holoenzyme. The oscillation of the RSUs was 9h out of phase relative to the transcripts. The amount of rbc transcripts was at its maximum in the dark while that of RSUs was at its maximum in the light phase suggesting that translation of the rbc transcripts is activated by light as previously hypothesized. A possible post-translational regulation that might be involved in the accumulation of a 37-kDa N-terminal LSU fragment during the light phase is discussed. BIOLOGICAL SIGNIFICANCE: A novel MS based approach enabling the exact stoichiometric analysis and absolute quantification of protein complexes is presented in this article. The application of this method revealed new insights in RuBisCO subunit dynamics.

System-level network analysis of nitrogen starvation and recovery in Chlamydomonas reinhardtii reveals potential new targets for increased lipid accumulation.

BACKGROUND: Nitrogen starvation is known to cause drastic alterations in physiology and metabolism leading to the accumulation of lipid bodies in many microalgae, and it thus presents an important alternative for biofuel production. However, despite the importance of this process, the molecular mechanisms that mediate the metabolic remodeling induced by N starvation and especially by stress recovery are still poorly understood, and new candidates for bioengineering are needed to make this process useful for biofuel production. RESULTS: We have studied the molecular changes involved in the adaptive mechanisms to N starvation and full recovery of the vegetative cells in the microalga Chlamydomonas reinhardtii during a four-day time course. High throughput mass spectrometry was employed to integrate the proteome and the metabolome with physiological changes. N starvation led to an accumulation of oil bodies and reduced Fv/Fm.. Distinct enzymes potentially participating in the carbon-concentrating mechanism (CAH7, CAH8, PEPC1) are strongly accumulated. The membrane composition is changed, as indicated by quantitative lipid profiles. A reprogramming of protein biosynthesis was observed by increased levels of cytosolic ribosomes, while chloroplastidic were dramatically reduced. Readdition of N led to, the identification of early responsive proteins mediating stress recovery, indicating their key role in regaining and sustaining normal vegetative growth. Analysis of the data with multivariate correlation analysis, Granger causality, and sparse partial least square (sPLS) provided a functional network perspective of the molecular processes. Cell growth and N metabolism were clearly linked by the branched chain amino acids, suggesting an important role in this stress. Lipid accumulation was also tightly correlated to the COP II protein, involved in vesicle and lysosome coating, and a major lipid droplet protein. This protein, together with other key proteins mediating signal transduction and adaption (BRI1, snRKs), constitute a series of new metabolic and regulatory targets. CONCLUSIONS: This work not only provides new insights and corrects previous models by analyzing a complex dataset, but also increases our biochemical understanding of the adaptive mechanisms to N starvation in Chlamydomonas, pointing to new bioengineering targets for increased lipid accumulation, a key step for a sustainable and profitable microalgae-based biofuel production.

The different proteomes of Chlamydomonas reinhardtii.

Protein identification and proteome mapping mostly rely on the combination of tandem mass spectrometry and sequence database searching. Despite constant improvements achieved in instrumentation, search algorithms, and genome annotations, little effort has been invested in estimating the impact of different genome annotation releases on the final results of a proteome study. We have used a large dataset of mass spectra obtained using an Orbitrap LTQ XL instrument, covering different growth situations of the model species Chlamydomonas reinhardtii. More than one million spectra were analyzed employing the SEQUEST algorithm and four different databases corresponding to the major Chlamydomonas genome assemblies. In total more than 3000 proteins and about 11,000 peptides were identified. 238 proteins were exclusively detected in assembly 3.0 in contrast to 1222 missing proteins only detectable in other databases. The comparison of the results demonstrates that the database selection affects not only the number of identified proteins but also label free quantitation and the biological interpretation of the results. Lists of protein accessions exclusively assigned to individual C. reinhardtii genome assemblies and annotations are provided as a resource for proteogenomic studies.

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

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.