A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin.
A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin.
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DOI：
10.1002/pmic.201400155
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发表时间：
2015-01
期刊：
影响因子：
3.4
通讯作者：
Gygi SP
中科院分区：
文献类型：
--
作者：
Paulo JA;Gygi SP
我们应用了一种多重的、基于质谱学的策略来询问三个酵母菌株在两种生长条件下的蛋白质组和磷蛋白质组。酵母蛋白Bmh1和Bmh2类似于14-3-3蛋白家族，具有广泛的细胞功能，包括调节磷酸化事件。类似地，雷帕霉素是一种可以调节磷酸化事件的药物。通过一系列TMT10-plex实验，以雷帕霉素和二甲基亚砜处理酿酒酵母野生型菌株和两株缺失菌株(BMH1Δ和BMH2Δ)为对照，研究了野生型和缺失菌株的蛋白质组和磷酸蛋白质组的变化。我们的3×3+1策略允许对三个菌株中的每一个进行三重分析，外加一个由所有样本的等量混合组成的额外样本。我们量化了4000多个蛋白质和20,000个磷酸化事件。其中，我们量化了所有20个样本中的3700多个蛋白质和每个药物治疗中的14,300多个磷酸化事件。总体而言，从四个TMT10-plex实验收集的数据需要在质谱仪上收集大约一周的数据。这项研究强调了Bmh1和Bmh2的复杂细胞作用以及对雷帕霉素治疗的反应，并强调了多重蛋白质组学技术在阐明综合蛋白质组和磷酸蛋白质组方面的作用。
We applied a multiplexed, mass spectrometry-based strategy to interrogate the proteome and phosphoproteome of three yeast strains under two growth conditions. The yeast proteins Bmh1 and Bmh2, analogs to the 14-3-3 protein family, have a wide-array of cellular functions including the regulation of phosphorylation events. Similarly, rapamycin is a drug that can regulate phosphorylation events. By performing a series of TMT10-plex experiments, we investigated the alterations in the proteome and phosphoproteome of wildtype and two deletion strains (bmh1Δ and bmh2Δ) of S. 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 TMT10-plex experiments required approximately one 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.