Proteomic analysis of eukaryotic protein synthesis

描述(申请人提供)：这项建议的长期目标是 识别和理解蛋白质翻译的复杂系统 它的分子相互作用。真核翻译的研究始于 20世纪70年代，使用传统的生化和遗传方法。我们最近做了 展示了一种基于使用全基因组的新的现代方法 序列和蛋白质组学可以为这一复杂和必要的问题提供新的线索 细胞过程。我开创了一种蛋白质组学方法，名为“直接 “大蛋白复合体分析”或DALPC直接鉴定新基因 与翻译复合体相关的蛋白质。这个高度敏感的团块 光谱学方法能够全面识别100个个体 复杂混合物中的蛋白质以纳克水平存在，不需要 凝胶电泳法分离蛋白质。这种方法已经 鉴定出40S核心核糖体亚基的一种新成分。从开始 酿酒酵母，我的策略是提纯翻译蛋白 并利用质谱法对蛋白质进行综合鉴定 基于光谱的方法。翻译复合体将使用 多种途径。第一，积极翻译核糖体亚基和 相关的翻译因子将通过蔗糖梯度分离 细胞裂解产物的分级。其次，将剥离翻译因素 用高盐洗涤活性核糖体并从核心核糖体中分离出来 通过离心法分离颗粒。最后，亲和力标记的翻译蛋白将 在非变性条件下纯化以分离出物理上相关的 蛋白质。对于所有三种方法，组件都将使用 DALPC方法。识别出的新蛋白质将用一系列 生物信息学工具和数据存储库，用于优先处理蛋白质 进一步的刻画。然后将使用遗传和生化实验 验证是否发现新的蛋白质与翻译蛋白共纯化 复合体实际上参与了翻译。具体而言，删除或 将获得条件突变株，并与同基因的野生型菌株进行比较。 蛋白质合成活性、多聚核糖体分布和 对蛋白质抑制剂的敏感性。这些实验的成功将 大大增加我们对蛋白质最重要的过程的了解 综合。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify and understand the complex system of protein translation in terms of its molecular interactions. Eukaryotic translation has been studied since the 1970s using traditional biochemical and genetic approaches. We have recently demonstrated that a new modern approach based on the use of complete genome sequences arid proteomics can shed new light on this complex and essential cellular process. I have pioneered a proteomics approach called "Direct Analysis of Large Protein Complexes" or DALPC to directly identify novel proteins associated with translational complexes. This highly sensitive mass spectrometry method is capable of comprehensively identifying 100 individual proteins in complex mixtures present at nanogram levels without the need for separating the proteins by gel electrophoresis. This approach has already identified a novel component of the 40S core ribosomal subunit. Starting with yeast Saccharomyces cerevisiae, my strategy is to purify translational protein complexes and to comprehensively identify the proteins using mass spectrometry-based methods. Translation complexes will be isolated using multiple approaches. First, actively translating nbosome subunits and associated translation factors will be separated by sucrose gradient fractionation of cell lysates. Second, translation factors will be stripped off active ribosomes using high salt washes and separated from the core ribosome particles by centrifugation. Finally, affinity-tagged translation proteins will be purified under non-denaturing conditions to isolate physically associated proteins. For all three approaches, the components will be identified using the DALPC approach. Novel proteins identified will be analyzed using a series of bioinformatics tools and data repositories to prioritize the proteins for further characterization. Genetic and biochemical experiments will then be used to validate whether novel proteins found co-purifying with translational complexes are actually involved in translation. Specifically, deletion or conditional mutants will be obtained and compared to isogenic wild type strains for changes in protein synthesis activity, polyribosome profiles, and sensitivity to protein inhibitors. The success of these experiments will greatly increase our knowledge of the all-important process of protein synthesis.