SENSITIVE GLOBAL PROFILING OF PROTEOLYSIS IN APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白水解酶在多种生物过程的调控中起着重要作用。其中一个这样的过程是细胞凋亡，其中至少280个蛋白质被已知进行蛋白质分解处理，作为导致细胞程序性死亡的生化事件的一部分。增加这一复杂性的是，凋亡反应的性质可能会因凋亡刺激和细胞类型的不同而不同，这种方式并不总是可以预测的。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。这项工作的目的是建立一种新的方法，用于分析复杂的生化混合物中的蛋白分解情况，该方法是灵敏的、稳健的和通用的。这种方法将基于使用一种工程化的多肽连接酶来选择性地标记细胞裂解产物中的蛋白质N末端。该标记将允许对N-端肽进行亲和纯化和浓缩，以便随后通过串联质谱仪进行测序。实验样品中存在的N末端和对照样品中不存在的N末端的鉴定将表明在已测序的氨基酸位点上发生了蛋白质降解事件。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。然后，该方法的成熟版本将被应用于研究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，并确定新的细胞凋亡蛋白分解靶点。这项工作将为细胞凋亡的生物学提供新的线索，并将建立一种新的蛋白质组学方法，该方法将被应用于生物学其他领域的蛋白质分解研究。加州大学旧金山分校的质谱学设施将提供这项工作成功所必需的质谱学仪器、软件和专业知识。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteolysis plays an important role in the regulation of diverse biological processes. One such process is apoptosis, in which at least 280 proteins are known to undergo proteolytic processing as part of the biochemical events leading to programmed cell death. Adding to this complexity, the nature of the apoptotic response can vary depending on the apoptotic stimulus and the cell type in a manner that cannot always be predicted. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations. The aim of this work is to establish a novel method for global profiling of proteolysis in complex biochemical mixtures that is sensitive, robust, and general. This method will be based on the use of an engineered peptide ligase to selectively label protein N-termini in cell lysates. The label will permit affinity purification and enrichment of N-terminal peptides for subsequent sequencing by tandem mass spectrometry. Identification of N-termini present in experimental samples and absent in control samples will be indicative of a proteolytic event at the sequenced amino acid site. 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 survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, and to identify new targets of proteolysis in apoptosis. This work will shed new light on the biology of apoptosis and will establish a novel proteomic method that will find application to the study of proteolysis in other areas of biology. The UCSF Mass Spectrometry Facility will provide the mass spectrometry instrumentation, software, and expertise essential for the success of this work.