Proteome-wide analysis of sumoylation

描述（由申请人提供）：SUMO等泛蛋白样蛋白（Ubls）对蛋白质的共价修饰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相关性。因此，全面了解蛋白质的Ubl修饰对于了解基本生物过程如何调节以及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sumoylated蛋白质在蛋白质组学规模。这将使我们能够在各种生物和医学相关的设置中检测sumoylation模式的变化。我们提出的策略是表达SUMO融合到串联亲和标签，并通过两步亲和纯化产生纯化的级分sumoylated蛋白。纯化的组分将通过多维液相色谱和串联质谱的组合进行分析[以生成sumoylation谱。这些谱在鉴定用SUMO修饰的蛋白质方面将是有价值的，但更重要的是，比较从不同生理或发育状态的细胞产生的谱将有助于鉴定重要的调节因子。第一个目标将使用酵母作为模型系统，以检测在发酵过程中sumoylation模式的定量变化。|不同的细胞周期阶段和细胞周期检查点停滞。目的2试图适应的方法从/东哺乳动物细胞，并定量分析SUMO-1修饰的变化，以响应检查点诱导的细胞周期停滞。这项建议的重点是确定sumoylation目标，但我们相信，所提出的策略可以应用于其他修饰的泛素样蛋白没有任何显着的变化，并将形成基础的各种Ubl修饰配置文件的生成。

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.