Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232, USA.
Proteomics. 2020 Apr;20(7):e1900177. doi: 10.1002/pmic.201900177. Epub 2020 Mar 3.
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 In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.
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氨基酸位点，我们针对先前通过在翻译起始、延伸、终止或与核糖体复合物相互作用中的遗传和/或功能作用而被认为与真核 mRNA 翻译有关的酵母基因，进行了复制 TAP-MudPIT 和对照实验。每个靶向酵母 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相结合，用于鉴定和定量共纯化的蛋白质。为了提高灵敏度并最小化虚假的、非特异性的相互作用，我们采用了一种新的交叉验证方法来识别最具统计学意义的蛋白质-蛋白质相互作用。使用本文中讨论的实验和计算策略，计算了先前描述的典型真核 mRNA 翻译起始、延伸和终止复合物的蛋白质组成。此外，还鉴定了 S. cerevisiae 的 mRNA 翻译蛋白和复合物的具有统计学意义的未发表的蛋白质相互作用和磷酸化位点。
