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Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

ABSTRACT: The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry-based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carbon source, in triplicate, thereby permitting subsequent statistical analyses. We quantified more than 4700 proteins across all nine samples; 1003 proteins demonstrated statistically significant differences in abundance in at least one condition. The majority of altered proteins were classified as functioning in metabolic processes and as having cellular origins of plasma membrane and mitochondria. In contrast, proteins remaining relatively unchanged in abundance included those having nucleic acid-related processes, such as transcription and RNA processing. In addition, the comprehensiveness of the data set enabled the analysis of subsets of functionally related proteins, such as phosphatases, kinases, and transcription factors. As a resource, these data can be mined further in efforts to understand better the roles of carbon source fermentation in yeast metabolic pathways and the alterations observed therein, potentially for industrial applications, such as biofuel feedstock production.

SUBMITTER: Paulo JA

PROVIDER: S-EPMC4710237 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

Paulo Joao A JA O'Connell Jeremy D JD Gaun Aleksandr A Gygi Steven P SP

Molecular biology of the cell 20150923 22

The global proteomic alterations in the budding yeast Saccharomyces cerevisiae due to differences in carbon sources can be comprehensively examined using mass spectrometry-based multiplexing strategies. In this study, we investigate changes in the S. cerevisiae proteome resulting from cultures grown in minimal media using galactose, glucose, or raffinose as the carbon source. We used a tandem mass tag 9-plex strategy to determine alterations in relative protein abundance due to a particular carb ...[more]

PMID: 26399295

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Project description:Saccharomyces cerevisiae and Schizosaccharomyces pombe are the most commonly studied yeast model systems, yet comparisons of global proteome remodeling between these yeast species are scarce. Here, we profile the proteomes of S. cerevisiae and S. pombe cultured with either glucose or pyruvate as the sole carbon source to define common and distinctive alterations in the protein landscape across species. In addition, we develop an updated streamlined-tandem mass tag (SL-TMT) strategy that substitutes chemical-based precipitation with more versatile bead-based protein aggregation method (SP3) prior to enzymatic digestion and TMT labeling. Our new workflow, SL-SP3-TMT, allow for near-complete proteome profiles in a single experiment for each species. The data reveal expected alterations in protein abundance and differences between species, highlighted complete canonical biochemical pathways, and provided insight into previously uncharacterized proteins. The techniques used herein, namely SL-SP3-TMT, can be applied to virtually any experiment aiming to study remodeling of the proteome using a high-throughput, comprehensive, yet streamlined mass spectrometry-based strategy. SIGNIFICANCE: Saccharomyces cerevisiae and Schizosaccharomyces pombe are single-celled eukaryotes that diverged from a common ancestor over a period of 100 million years, such that evolution has driven fundamental differences between the two species. Cellular metabolism and the regulation thereof are vital for living organisms. Here, we hypothesize that large scale proteomic alterations are prevalent upon the substitution of glucose with another carbon source, in this case pyruvate. To efficiently process our samples, we developed an updated streamlined-tandem mass tag (SL-TMT) strategy with more versatile bead-based protein aggregation. The data revealed expected alterations in protein abundance and illustrated differences between species. We highlighted complete canonical biochemical pathways and provided insight into previously uncharacterized proteins.

Project description:BackgroundControl effective flux (CEF) of a reaction is the weighted sum of all fluxes through that reaction, derived from elementary flux modes (EFM) of a metabolic network. Change in CEFs under different environmental conditions has earlier been proven to be correlated with the corresponding changes in the transcriptome. Here we use this to investigate the degree of transcriptional regulation of fluxes in the metabolism of Saccharomyces cerevisiae. We do this by quantifying correlations between changes in CEFs and changes in transcript levels for shifts in carbon source, i.e. between the fermentative carbon source glucose and nonfermentative carbon sources like ethanol, acetate, and lactate. The CEF analysis is based on a simple stoichiometric model that includes reactions of the central carbon metabolism and the amino acid metabolism.ResultsThe effect of the carbon shift on the metabolic fluxes was investigated for both batch and chemostat cultures. For growth on glucose in batch (respiro-fermentative) cultures, EFMs with no by-product formation were removed from the analysis of the CEFs, whereas those including any by-products (ethanol, glycerol, acetate, succinate) were omitted in the analysis of growth on glucose in chemostat (respiratory) cultures. This resulted in improved correlations between CEF changes and transcript levels. A regression correlation coefficient of 0.60 was obtained between CEF changes and gene expression changes in the central carbon metabolism for the analysis of 5 different perturbations. Out of 45 data points there were no more than 6 data points deviating from the correlation. Additionally, up- or down-regulation of at least 75% of the genes were in qualitative agreement with the CEF changes for all perturbations studied.ConclusionThe analysis indicates that changes in carbon source are associated with a high degree of hierarchical regulation of metabolic fluxes in the central carbon metabolism as the change in fluxes are correlating directly with the change in transcript levels of genes encoding their corresponding enzymes. For amino acid biosynthesis there was, however, not found to exist a similar correlation, and this may point to either post-transcriptional and/or metabolic regulation, or be due to the absence of a direct perturbation on the amino acid pathways in these experiments.

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Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

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Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae.

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