Proteome-wide identification of protease targets

描述（由申请人提供）：本提案中描述的研究重点是开发一种基于质谱（MS）的技术，该技术旨在实现蛋白酶底物的蛋白质组范围分析。蛋白酶在信号转导途径中发挥着重要作用，在细胞凋亡、细胞周期、肿瘤和分化等一系列生理过程中发挥着重要作用。尽管蛋白酶在信号转导途径中的重要性，但目前还没有确定蛋白酶靶点或确定细胞信号通路中蛋白水解加工事件发生的一般方法。我们开发了新的化学方法和试剂，使我们能够用固相同位素编码标签在蛋白质的n端专门标记蛋白质。本提案旨在发展和扩展这些技术，以开发一种高通量的基于质谱的蛋白质组学技术，能够表征细胞中的蛋白质水解过程。该方法基于这样一种思想，即在对给定蛋白质进行蛋白水解后，至少会产生两种产物，其中一种产物具有与亲本蛋白相同的n端，而另一种产物具有仅在蛋白水解时暴露的新n端。因此，基于质谱的蛋白质n端多肽分析将提供一种检测和鉴定蛋白质水解处理蛋白的方法。为了发展这一技术，本提案的具体目标是：(1)优化n端肽（MNP）的质谱过程；(2)应用MNP程序鉴定hiv相关淋巴瘤细胞系中caspase-3和一氧化氮（NO）调节的蛋白水解的底物。总之，本提案中描述的实验将导致新技术的发展，我们期望该技术将成为分析细胞信号中蛋白酶的基本工具。

DESCRIPTION (provided by applicant): The research described in this proposal is focused on the development of a mass spectrometry (MS)-based technology that is designed to enable proteome-wide profiling of protease substrates. Proteases play a fundamental role in signal transduction pathways and have roles in a wide array of physiologic processes such as apoptosis, cell cycle, cancer, and differentiation. Despite the importance of proteases in signal transduction pathways, general methods to identify the targets of proteases or to identify the occurrence of proteolytic processing events in cellular signaling pathways are not available. We have developed novel chemical procedures and reagents that enable us to label proteins exclusively on their N-terminus with solid- phase, isotope-coded tags. This proposal seeks to develop and expand these techniques to develop a high- throughput MS-based proteomic technology capable of characterizing proteolytic processing in cells. The method relies on the idea that following proteolysis of a given protein, at least two products are generated, one of which has an N-terminus that is identical to the parent protein, while the other has a new N-terminus that is exposed only upon proteolysis. Thus, MS-based profiling of peptides derived from the N-terminus of proteins would provide a method to detect and identify proteolytically processed proteins. In order to develop this technology, the specific aims of this proposal are: (1) To optimize the MS of N-terminal peptides (MNP) procedure; and (2) To apply the MNP procedure to identify substrates of caspase-3 and nitric oxide (NO)- regulated proteolysis in HIV-associated lymphoma cell lines. Together, the experiments described in this proposal will result in the generation of development of a novel technology that we expect will become a fundamental tool for the analysis of proteases in cellular signaling.