To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

中文翻译：

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在真核mRNA翻译中蛋白质相互作用的靶向鉴定。

为了鉴定真核mRNA翻译中的蛋白质-蛋白质相互作用和磷酸化的氨基酸位点，针对先前涉及真核mRNA翻译的酿酒酵母基因，通过其在翻译起始，延伸，终止中的遗传和/或功能作用，进行复制TAP-MudPIT和对照实验，或与核糖体复合物的相互作用。每个目标酵母TAP标记的mRNA翻译蛋白的重复串联亲和纯化与多维液相色谱和串联质谱分析一起用于鉴定和定量共纯化蛋白。为了提高灵敏度并最大程度地减少虚假的非特异性相互作用，采用了一种新颖的交叉验证方法来识别统计学上最有意义的蛋白-蛋白相互作用。使用本文讨论的实验和计算策略，可以计算出前述真核mRNA翻译起始，延伸和终止复合物的蛋白质组成。另外，鉴定了酿酒酵母的mRNA翻译蛋白和复合物的统计学上显着的未公开的蛋白相互作用和磷酸化位点。