Sensitive Global Profiling of Proteolysis in Apoptosis

描述(申请人提供)：蛋白质分解在调节不同的生物过程中起着重要作用。不幸的是，目前监测复杂样品中蛋白质分解事件的方法受到严重限制。这项建议的目的是建立一种新的方法，用于对生化混合物中的蛋白质分解进行全球图谱分析，该方法灵敏、健壮和通用。这种方法的基础是检测新形成的蛋白质N-末端。这将使用枯草杆菌酶来完成，枯草杆菌酶是一种被设计成作为一种有效的肽连接酶发挥功能的蛋白酶的变种。在复杂的生化混合物中，枯草杆菌酶将被用来选择性地将标记的多肽连接到蛋白水解蛋白的N-末端。该标记将允许亲和纯化和浓缩连接的蛋白质，以便随后通过串联质谱仪进行鉴定。首先，细胞凋亡中的蛋白分解分析将被用作方法开发的模型系统。该方法的成熟版本将被应用于：a)调查不同刺激和不同细胞类型引起的细胞凋亡中蛋白分解模式的多样性，以及b)确定细胞凋亡中蛋白分解的新靶点，特别是为化疗干预寻找新的靶点。

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.