Proteome-wide analysis of sumoylation

描述（由申请人提供）：SUMO等泛素样蛋白（ubitin - likeprotein, Ubls）对蛋白质的共价修饰已被认为是大多数生物通路的重要调控机制，其中许多具有重要的医学意义。因此，全面了解蛋白质的ubl修饰对于理解基本生物过程是如何被调节的以及找到治疗各种疾病的新策略至关重要。为了达到这一目标，我们建议开发和应用工具来分离和鉴定在蛋白质组学尺度上的聚合蛋白。这将使我们能够在各种生物和医学相关环境中检测到sumoylation模式的变化。我们提出的策略是将SUMO融合到串联亲和标签上，并通过两步亲和纯化产生纯化的SUMO化蛋白。纯化后的馏分将通过多维液相色谱和串联质谱相结合的方法进行分析，以生成sumo酰化谱。这样的基因图谱对于鉴定被SUMO修饰的蛋白质是有价值的，但更重要的是，比较不同生理或发育状态下细胞产生的基因图谱将有助于识别重要的调节因子。第一个目标将使用酵母作为模型系统来检测在不同细胞周期阶段和细胞周期检查点阻滞期间sumoylation模式的定量变化。目的2试图将该方法应用于哺乳动物细胞，并定量分析在检查点诱导的细胞周期阻滞下SUMO-1修饰的变化。该建议的重点是识别sumoylation靶点，但我们相信，该策略可以应用于泛素样蛋白的其他修饰，而不会产生任何重大变化，并将为生成各种Ubl修饰谱奠定基础。

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.