Proteome-wide identification of protease targets

蛋白酶靶标的蛋白质组范围鉴定

• 批准号: 7062593
• 负责人: SAMIE R JAFFREY
• 金额: $ 21万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062593
• 关键词: biological signal transduction; biomarker; biotechnology; cell line; endopeptidases; high throughput technology; mass spectrometry; nitric oxide; protein localization; protein structure function; proteolysis; proteomics; stable isotope; technology /technique development; tissue /cell culture

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.

描述（由申请人提供）：本提案中描述的研究重点是开发基于质谱（MS）的技术，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起着重要作用，并在细胞凋亡、细胞周期、癌症和分化等广泛的生理过程中发挥作用。尽管蛋白酶在信号转导途径中的重要性，但鉴定蛋白酶的靶标或鉴定细胞信号转导途径中蛋白水解加工事件的发生的一般方法不可用。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的标签仅在蛋白质的N-末端标记蛋白质。该提议寻求开发和扩展这些技术，以开发能够表征细胞中蛋白水解加工的高通量基于MS的蛋白质组学技术。该方法依赖于这样的想法，即在给定蛋白质的蛋白水解之后，产生至少两种产物，其中一种具有与亲本蛋白质相同的N-末端，而另一种具有仅在蛋白水解时暴露的新的N-末端。因此，基于MS的蛋白质N端肽谱分析将提供一种检测和鉴定蛋白水解加工蛋白质的方法。为了发展这一技术，本研究的具体目标是：（1）优化N-末端肽（MNP）的MS方法;（2）应用MNP方法鉴定HIV相关淋巴瘤细胞系中caspase-3和一氧化氮（NO）调节的蛋白水解底物。总之，本提案中描述的实验将导致一种新技术的开发，我们预计该技术将成为分析细胞信号中蛋白酶的基本工具。