Proteome-wide analysis of sumoylation

sumoylation 的全蛋白质组分析

• 批准号: 7030823
• 负责人: Peter Kaiser
• 金额: $ 17.39万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7030823
• 关键词: cell cycle; proteins; yeasts

DESCRIPTION (provided by applicant): Covalent modification of proteins by ubiquitin-like-proteins (Ubls) such as SUMO has been recognized as an important regulatory mechanism for most biological pathways, many of which are of great medical relevance. A comprehensive knowledge of Ubl-modification of proteins is therefore critical to understand how fundamental biological processes are regulated and to find new strategies for the treatment of a variety of diseases. To reach this goal, we propose to develop and apply tools to isolate and identify sumoylated proteins jn a proteomic scale. This will allow us to detect changes in sumoylation patterns in a variety of biological and medically relevant settings. The strategy we propose is to express SUMO fused to a tandem affinity-tag and generate a purified fraction of sumoylated proteins by a two-step affinity purification. The purified fractions will be analyzed by a combination of multidimensional liquid chromatography and tandem mass spectrometry [to generate sumoylation profiles. Such profiles will be valuable in identification of proteins that are modified with SUMO but more importantly, comparison of profiles generated from cells in different physiological or developmental states will help to identify important regulatory factors. The first aims will use yeast as a model system to detect quantitative changes in sumoylation patterns during |the different cell cycle stages and a cell cycle checkpoint arrest. Aim 2 attempts to adapt the approach from /east to mammalian cells and to quantitatively analyze changes in SUMO-1 modifications in response to a checkpoint-induced cell cycle arrest. This proposal is focused on identification of sumoylation targets, but we believe that the proposed strategy can applied to other modifications with ubiquitin-like proteins without any significant changes and will form the foundation for the generation of a variety of Ubl modification profiles.

描述(申请人提供)：泛素样蛋白(UBL)对蛋白质的共价修饰，如SUMO，已被认为是大多数生物途径的重要调节机制，其中许多具有重要的医学意义。因此，全面了解蛋白质的Ubl修饰对于了解基本的生物过程是如何调节的并找到治疗各种疾病的新策略至关重要。为了实现这一目标，我们建议开发和应用工具来分离和鉴定蛋白质组学水平上的总合作用蛋白。这将使我们能够在各种生物和医学相关环境中检测相加模式的变化。我们提出的策略是将相扑融合到串联亲和标签上表达，并通过两步亲和纯化产生纯化的相扑蛋白部分。提纯的馏分将通过多维液相色谱和串联质谱仪的组合进行分析，以生成苏莫化图谱。这些图谱将在鉴定被相扑修饰的蛋白质方面很有价值，但更重要的是，比较处于不同生理或发育状态的细胞产生的图谱将有助于识别重要的调控因素。第一个目标将使用酵母作为一个模型系统，以检测不同细胞周期阶段和细胞周期检查点停滞期间苏莫化模式的数量变化。目的2尝试使该方法适用于哺乳动物细胞，并定量分析相扑-1修饰在检查点诱导的细胞周期停滞反应中的变化。这一建议的重点是识别苏莫化靶标，但我们相信，所提出的策略可以应用于其他泛素样蛋白的修饰，而不会有任何显著的变化，并将为产生各种Ub1修饰图谱奠定基础。