Sensitive Global Profiling of Proteolysis in Apoptosis

描述（由申请人提供）：蛋白质水解在多种生物过程的调节中起着重要作用。不幸的是，目前监测复杂样品中蛋白水解事件的方法存在严重的局限性。本提案的目的是建立一种新的方法来分析生化混合物中的蛋白质水解，这是敏感的，稳健的和通用的。该方法的基础将是检测新形成的蛋白n端。这将使用枯草菌酶来完成，枯草菌酶是蛋白酶枯草菌素的一种变体，已被设计成一种有效的肽连接酶。枯草化酶将用于在复杂的生化混合物中选择性地将标记肽连接到蛋白水解蛋白的n端。该标签将允许亲和纯化和富集连接蛋白，以便随后通过串联质谱进行鉴定。细胞凋亡中蛋白质水解的分析将首先用作方法开发的模型系统。然后，该方法的成熟版本将应用于：A)调查不同刺激和不同细胞类型引起的细胞凋亡中蛋白质水解模式的多样性，以及b)确定细胞凋亡中蛋白质水解的新靶点，特别强调确定化疗干预的新点。

DESCRIPTION (provided by applicant): Proteolysis plays an important role in the regulation of diverse biological processes. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations. The aim of this proposal is to establish a novel method for global profiling of proteolysis in biochemical mixtures that is sensitive, robust, and general. The basis for this method will be to detect newly formed protein N-termini. This will be accomplished using subtiligase, a variant of the protease subtilisin that has been engineered to function as an efficient peptide ligase. Subtiligase will be employed to selectively ligate a labeled peptide onto N-termini of proteolyzed proteins in complex biochemical mixtures. The label will allow affinity purification and enrichment of ligated proteins for subsequent identification by tandem mass spectrometry. Analysis of proteolysis in apoptosis will at first be used as a model system for method development. A matured version of the method will then be applied to: a) survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, and b) identify new targets of proteolysis in apoptosis, with particular emphasis on identification of new points for chemotherapeutic intervention.