Sensitive Global Profiling of Proteolysis in Apoptosis

蛋白质水解在调节多种生物过程中起着重要作用。不幸的是，目前 用于监测复杂样品中蛋白水解事件的方法受到严重的限制。的目的 建议建立一种用于生物化学混合物中蛋白质水解的全局分析的新方法， 灵敏、健壮和通用。该方法的基础是检测新形成的蛋白质N-末端。 这将使用枯草杆菌蛋白酶来实现，枯草杆菌蛋白酶是蛋白酶枯草杆菌蛋白酶的变体，其已被工程化以 作为有效的肽连接酶发挥作用。将使用枯草杆菌连接酶选择性地连接标记的肽 在复杂的生化混合物中蛋白水解蛋白的N-末端上。标签将允许亲和力 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。一 成熟的版本的方法，然后将被应用于：a）调查的多样性蛋白水解模式， 由不同刺激物和在不同细胞类型中引起的细胞凋亡，和B）鉴定细胞凋亡中蛋白水解的新靶点， 细胞凋亡，特别强调识别化疗干预的新点。

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.