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. 2015 Jan;15(2-3):474-86.
doi: 10.1002/pmic.201400155. Epub 2014 Dec 17.

A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin

Affiliations

A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin

Joao A Paulo et al. Proteomics. 2015 Jan.

Abstract

We applied a multiplexed, MS-based strategy to interrogate the proteome and phosphoproteome of three yeast strains under two growth conditions in triplicate. The yeast proteins brain modulosignalin homologue (Bmh)1 and Bmh2, analogs to the 14-3-3 protein family, have a wide array of cellular functions including the regulation of phosphorylation events. Moreover, rapamycin is a drug that can regulate phosphorylation events. By performing a series of tandem mass tag 10-plex experiments, we investigated the alterations in the proteome and phosphoproteome of wildtype and two deletion strains (bmh1Δ and bmh2Δ) of Saccharomyces cerevisiae treated with rapamycin and DMSO as a control. Our 3 × 3 + 1 strategy allowed for triplicate analysis of each of the three strains, plus an additional sample consisting of an equal mix of all samples. We quantified over 4000 proteins and 20,000 phosphorylation events. Of these, we quantified over 3700 proteins across all 20 samples and over 14,300 phosphorylation events within each drug treatment. In total, data collected from four tandem mass tag 10-plex experiments required approximately 1 week of data collection on the mass spectrometer. This study underscores the complex cellular roles of Bmh1 and Bmh2 coupled with response to rapamycin treatment and emphasizes the utility of multiplexed proteomic techniques to elucidate comprehensive proteomes and phosphoproteomes.

Keywords: 14-3-3; Bmh; Cell biology; Multiplexing; Rapamycin; Yeast.

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Conflict of interest statement

Conflicts of interest

The authors acknowledge no conflict of interest.

Figures

Figure 1
Figure 1. Experimental overview
A) Three yeast strains (wildtype, bmh1Δ and bmh2Δ) were grown in biological triplicate. Cultures were split so that half was treated with rapamycin and the other half with DMSO as a control. Mass spectrometric analysis was performed at the protein and phosphopeptide levels. B) Representation of the 18 samples (plus two mixed samples) that were used for each analysis. Illustrated is the experimental set-up for both the protein and phosphosite level experiments.
Figure 2
Figure 2. Clustering diagrams of proteins with statistically significant differences in abundance
A) Proteins in the DMSO-treated control cultures with examples of those enriched in select KEGG pathways. We highlighted two proteins that were up-regulated in gluconeogenesis/glycolysis and down-regulated in metal ion binding. B) Proteins in the rapamycin-treated cultures. We highlighted two proteins that were up-regulated in oxidative phosphorylation and down-regulated in vitamin biosynthesis. The color scale of the heat map was capped at +/−2 and represented log2(mutant/wildtype). * indicates statistical significance as demonstrated by a Bonferroni-corrected p-value <0.01.
Figure 3
Figure 3. Clustering diagrams of phosphosites with statistically significant differences in abundance
A) Phosphosites in the DMSO-treated control cultures with examples of those enriched in certain KEGG pathways. We highlighted two phosphorylation sites that were up-regulated with RRxS sites and down-regulated with SP sites. B) Phosphosites in the rapamycin-treated control cultures. We highlighted two phosphorylation sites that were up-regulated with RRxS sites and down-regulated with SP sites. The color scale of the heat map was capped at +/−3 and represented log2(mutant/wildtype). * indicates statistical significance as demonstrated by a Bonferroni-corrected p-value <0.01.
Figure 4
Figure 4. Global alterations in proteome and phosphoproteome of wildtype, bmh1Δ and bmh2Δ yeast strains resulting from rapamycin treatment
The histograms were smoothed using the probability density function and showed log2 fold change for rapamycin-to-DMSO treatment at the A) protein and B) phosphosite levels.

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