Genomics

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Quantitative proteomics of anaerobic and aerobic yeast cultures


ABSTRACT: Saccharomyces cerevisiae is unique among yeasts for its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of the S. cerevisiae to anaerobiosis was investigated using metabolic stable isotope labeling in aerobic and anaerobic glucose-limited chemostat cultures, followed by proteome analysis to relatively quantify protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many responses of S. cerevisiae to oxygen availability were hitherto unreported. Comparison of transcriptome and proteome from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl tRNA synthesis, purine-nucleotide synthesis and amino-acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights in the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the messenger RNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology. Protein levels versus transcript level: Systematic analysis of the control levels at which the yeast response to anaerobiosis takes place was performed using previously published transcript data obtained from yeast cultures grown under strictly identical conditions as described for the current proteome analysis. Affymetrix microarrays from five aerobic and four anaerobic independent culture replicates were used for this analysis. These comparison data are summarized in the table below. These array data are publicly available at the gene expression repository Gene Expression Omnibus under accession number GSE4804. Keywords: proteomic, nanoflow-LC-MS/MS Overall design: The relative protein levels of anaerobically versus aerobically grown S. cerevisiae were investigated using metabolic stable isotope labeling. The biomass was produced using carbon limited chemostat cultures. Each biomass was produced twice, once using 14N labeled nitrogen source (ammonium sulfate) and once using 98% 15N nitrogen source. Subsequently, biomass was harvested, and protein extracts were prepared. Two mixed samples were prepared from these four protein samples, containing equal protein content of 14N aerobic and 15N anaerobic cultures for the first sample and equal protein content of 15N aerobic and 14N anaerobic cultures for the second sample. The two resulting samples were separated on an SDS-PAGE gel, and each lane was equally fragmented into 40 slices resulting in sub-samples based on protein size. This was followed by an in-gel protein digestion using trypsin, and analysis in triplicate by nanoflow-LC-MS/MS. Proteins were identified using MS/MS, and the relative protein levels were obtained by comparing the extracted ion chromatograms of the co-eluting 14N and 15N labeled peptide pairs

INSTRUMENT(S): Quantitative proteomics of anaerobic and aerobic yeast cultures

SUBMITTER: Marco J.L de Groot  

PROVIDER: GSE7365 | GEO | 2007-09-26

SECONDARY ACCESSION(S): PRJNA100439

REPOSITORIES: GEO

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Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals post-transcriptional regulation of key cellular processes.

de Groot Marco J L MJ   Daran-Lapujade Pascale P   van Breukelen Bas B   Knijnenburg Theo A TA   de Hulster Erik A F EA   Reinders Marcel J T MJ   Pronk Jack T JT   Heck Albert J R AJ   Slijper Monique M  

Microbiology (Reading, England) 20071101 Pt 11


Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic respon  ...[more]

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