Proteomic analysis of eukaryotic protein synthesis

真核蛋白质合成的蛋白质组学分析

• 批准号: 6620903
• 负责人: ANDREW J. LINK
• 金额: $ 30.96万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620903
• 关键词: Saccharomyces cerevisiae; chromatography; eukaryote; functional /structural genomics; fungal genetics; fungal proteins; gene environment interaction; gene expression; gene mutation; genetic regulation; genetic regulatory element; genetic translation; intermolecular interaction; mass spectrometry; molecular assembly /self assembly; northern blottings; polysomes; posttranslational modifications; protein biosynthesis; protein purification; protein structure function; proteomics; reporter genes; ribosomal proteins; western blottings; yeast two hybrid system

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.

描述（由申请人提供）：本提案的长期目标是 识别和理解蛋白质翻译的复杂系统， 分子间的相互作用真核生物翻译的研究始于1970年， 使用传统的生物化学和遗传学方法。我们最近 证明了一种新的现代方法， 序列和蛋白质组学可以揭示这一复杂而重要的 细胞过程我开创了一种蛋白质组学方法， 大蛋白质复合物分析”或DALPC直接鉴定新的 与翻译复合物相关的蛋白质。这个高度敏感的肿块 光谱法能够全面识别100个个体 蛋白质在复杂的混合物中以纳克水平存在，而不需要 通过凝胶电泳分离蛋白质。这种做法已经 鉴定了40S核心核糖体亚基的新组分。首先是 酵母酿酒酵母，我的策略是纯化翻译蛋白 复合物，并使用质量 基于光谱的方法。翻译复合体将被隔离使用 多种方法。首先，主动翻译核糖体亚基， 相关的翻译因子将通过蔗糖梯度分离 细胞裂解物的分级分离。其次，翻译因素将被剥离 活性核糖体使用高盐洗涤并与核心核糖体分离 颗粒离心。最后，亲和标记的翻译蛋白将 在非变性条件下纯化以分离物理相关的 proteins.对于所有三种方法，将使用 DALPC方法。新的蛋白质鉴定将使用一系列的 生物信息学工具和数据库，以优先考虑蛋白质， 进一步表征。然后将使用遗传和生物化学实验 为了验证是否发现与翻译共纯化的新蛋白质， 复合体实际上参与了翻译。具体而言，删除或 将获得条件突变体并与同基因野生型菌株进行比较 蛋白质合成活性、多聚核糖体特征的变化， 对蛋白质抑制剂敏感。这些实验的成功将 大大增加了我们对蛋白质重要过程的了解 合成.