Quantitative Analysis of the Cellular Lipidome of <em>Saccharomyces Cerevisiae</em> Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Quantitative Metabolomics of <em>Saccharomyces Cerevisiae</em> Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Concordia University

Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses. Metabolic labeling allows cells from different experiments to be mixed prior to analysis. This approach has been reported for prokaryotic cells. Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae. Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins. D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000). Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media. Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S. cerevisiae. Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.

Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine.

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H(2)O(2) to H(2)O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Delta) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H(2)O(2). Transformation of ccp1Delta with ccp1(W191F), which encodes the CCP(W191F) mutant enzyme lacking CCP activity, significantly increased the sensitivity to H(2)O(2) of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Delta-ccp1(W191F) exhibited wild-type tolerance to H(2)O(2), which exceeded that of ccp1Delta. Challenge with H(2)O(2) caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Delta exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H(2)O(2) exposure in ccp1Delta than in ccp1Delta-ccp1(W191F) and wild-type cells. The phenotypic differences reported here between the ccp1Delta and ccp1Delta-ccp1(W191F) strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.

Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling.

An efficient D10-Leu metabolic-labeling method combined with isotope-ratio quantitation by MALDI-TOF MS was used to probe the response of the yeast proteome to H2O2. Control cultures correct for effects not associated with H2O2 challenge. A stress-response index to H2O2 (SRIH2O2) is defined, and values are reported for seven proteins at 45-225 min following exposure to 0.4 mM H2O2. The time course of protein accumulation in unstressed cells following the H10- to D10-SCD switch suggests that proteome responses at <45 min could be monitored by addition of excess D10-Leu to H10-cultures.

Evaluation of D10-Leu metabolic labeling coupled with MALDI-MS analysis in studying the response of the yeast proteome to H2O2 challenge.

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q(10) (CoQ(10)), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ(10) precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ(10) was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ(10)-producing E. coli strain resulted in an increase in CoQ(10) content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ(10) content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10).

Identifying the genetic basis of complex microbial phenotypes is currently a major barrier to our understanding of multigenic traits and our ability to rationally design biocatalysts with highly specific attributes for the biotechnology industry. Here, we demonstrate that strain evolution by meiotic recombination-based genome shuffling coupled with deep sequencing can be used to deconstruct complex phenotypes and explore the nature of multigenic traits, while providing concrete targets for strain development.

Deconstructing the genetic basis of spent sulphite liquor tolerance using deep sequencing of genome-shuffled yeast.

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.

Heng Jiang

Department of Chemistry and Biochemistry,
Centre for Biological Applications of Mass Spectrometry,
Department of Chemistry and Biochemistry, Centre for Biological Applications of Mass Spectrometry

Concordia University

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Heng Jiang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry and Biochemistry,Centre for Biological Applications of Mass Spectrometry,Department of Chemistry and Biochemistry, Centre for Biological Applications of Mass Spectrometry

Concordia University

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Department of Chemistry and Biochemistry,
Centre for Biological Applications of Mass Spectrometry,
Department of Chemistry and Biochemistry, Centre for Biological Applications of Mass Spectrometry