Sensitive Global Profiling of Proteolysis in Apoptosis

描述（由申请人提供）：蛋白水解在多种生物过程的调节中起着重要作用。不幸的是，目前用于监测复杂样品中的蛋白水解事件的方法受到严重的限制。本提案的目的是建立一种新的方法，全球分析生化混合物中的蛋白质水解，是敏感的，强大的，和一般的。该方法的基础是检测新形成的蛋白质N-末端。这将使用枯草杆菌连接酶来实现，枯草杆菌连接酶是蛋白酶枯草杆菌蛋白酶的变体，其已被工程化以用作有效的肽连接酶。枯草杆菌酶将用于选择性地将标记的肽连接到复杂的生物化学混合物中的蛋白水解蛋白的N-末端上。标记将允许亲和纯化和富集连接的蛋白质，用于随后通过串联质谱法鉴定。细胞凋亡中蛋白水解的分析将首先用作方法开发的模型系统。然后将该方法的成熟版本应用于：a）调查由不同刺激和不同细胞类型引起的细胞凋亡中蛋白水解模式的多样性，和B）鉴定细胞凋亡中蛋白水解的新靶点，特别强调鉴定用于化疗干预的新点。

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.