de Godoy, L. M. F. et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, 1251-1254

Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Nature (Impact Factor: 41.46). 10/2008; 455(7217):1251-4. DOI: 10.1038/nature07341
Source: PubMed


Mass spectrometry is a powerful technology for the analysis of large numbers of endogenous proteins. However, the analytical challenges associated with comprehensive identification and relative quantification of cellular proteomes have so far appeared to be insurmountable. Here, using advances in computational proteomics, instrument performance and sample preparation strategies, we compare protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spans more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins. Stable-isotope labelling by amino acids in cell culture (SILAC) quantification was very accurate across the proteome, as demonstrated by one-to-one ratios of most yeast proteins. Key members of the pheromone pathway were specific to haploid yeast but others were unaltered, suggesting an efficient control mechanism of the mating response. Several retrotransposon-associated proteins were specific to haploid yeast. Gene ontology analysis pinpointed a significant change for cell wall components in agreement with geometrical considerations: diploid cells have twice the volume but not twice the surface area of haploid cells. Transcriptome levels agreed poorly with proteome changes overall. However, after filtering out low confidence microarray measurements, messenger RNA changes and SILAC ratios correlated very well for pheromone pathway components. Systems-wide, precise quantification directly at the protein level opens up new perspectives in post-genomics and systems biology.

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Available from: Jesper V Olsen, Feb 05, 2014
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    • "Several large-scale quantitative phosphoproteomics studies conducted on yeast cells have already described changes in protein phosphorylation in response to different stimuli including pheromone (Gruhler et al, 2005), DNA damage (Smolka et al, 2007), fatty acid and peroxisome induction (Saleem et al, 2010) and kinase inhibition (Bodenmiller et al, 2010). Other large-scale studies using stable isotope labeling in cell culture (SILAC) highlighted the regulation of protein phosphorylation in yeast during the cell cycle (Holt et al, 2009), and as a function of ploidy (de Godoy et al, 2008). While MS-based phosphoproteomics enables the profiling of thousands of phosphopeptides from microgram amounts of sample, the identification of the phosphosites that are specific to the stimulus remains a challenge, due to the prevalence of promiscuous phosphorylation events arising from random encounters of kinases with abundant neighboring proteins (Levy et al, 2012). "
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    ABSTRACT: The ability of cells and organisms to survive and function through changes in temperature evolved from their specific adaptations to nonoptimal growth conditions. Responses to elevated temperatures have been studied in yeast and other model organisms using transcriptome profiling and provided valuable biological insights on molecular mechanisms involved in stress tolerance and adaptation to adverse environment. In contrast, little is known about rapid signaling events associated with changes in temperature. To gain a better understanding of global changes in protein phosphorylation in response to heat and cold, we developed a high temporal resolution phosphoproteomics protocol to study cell signaling in Saccharomyces cerevisiae. The method allowed for quantitative analysis of phosphodynamics on 2,777 phosphosites from 1,228 proteins. The correlation of kinetic profiles between kinases and their substrates provided a predictive tool to identify new putative substrates for kinases such as Cdc28 and PKA. Cell cycle analyses revealed that the increased phosphorylation of Cdc28 at its inhibitory site Y19 during heat shock is an adaptive response that delays cell cycle progression under stress conditions. The cellular responses to heat and cold were associated with extensive changes in phosphorylation on proteins implicated in transcription, protein folding and degradation, cell cycle regulation and morphogenesis. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    Molecular Systems Biology 06/2015; 11(6). DOI:10.15252/msb.20156170 · 10.87 Impact Factor
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    • "The yeast transcriptome has been studied in numerous conditions using microarrays and RNA-Sequencing (RNA-Seq) [9] [10] [11] [12]. Furthermore, protein-profiling experiments detected nearly all theoretically predicted yeast proteins in pre-fractionated extracts from growing cells [13] [14]. Very recent feasibility studies reported vastly improved experimental protocols yielding quantitative information about protein concentrations in total cell extracts [15] [16] [17]. "
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    Journal of Proteomics 02/2015; 119. DOI:10.1016/j.jprot.2015.01.015 · 3.89 Impact Factor
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    • "Rapid developments and technological advances in the field of mass spectrometry have made electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) a powerful tool for the analysis of peptides and proteins [1]–[2] with the capability to routinely identify sequences of tens of thousands of proteins [3]–[4]. Most analyses were performed using positive ion mode because the ionization of peptides and proteins in negative ion mode is relatively limited [5]. "
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