SENSITIVE GLOBAL PROFILING OF PROTEOLYSIS IN COMPLEX BIOCHEMICAL MIXTURES

复杂生化混合物中蛋白质水解的灵敏全局分析

• 批准号: 7724207
• 负责人: JAMES A WELLS
• 金额: $ 1.03万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724207
• 关键词: Affinity Chromatography; Amino Acid Sequence; Apoptosis; Area; Biochemical; Biological; Biological Process; Biology; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer software; Development; Disease; Engineering; Event; Funding; Grant; Health; Human; Inflammation; Institution; Label; Ligase; Light; Mass Spectrum Analysis; Methods; Monitor; N-terminal; Peptides; Phosphorylation; Play; Post-Translational Protein Processing; Proteins; Proteolysis; Proteomics; Regulation; Research; Research Personnel; Resources; Role; Sampling; Serum; Site; Source; System; Ubiquitination; United States National Institutes of Health; Work; base; instrumentation; novel; success; tandem mass spectrometry

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. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations and lag far behind those typically used for proteomic profiling of other post-translational modifications such as phosphorylation and ubiquitination. The aim of our work is development and application of a novel method for global profiling of proteolysis in complex biochemical mixtures that is sensitive, robust, and general. This method is based on the use of an engineered peptide ligase to selectively label protein N-termini in cell lysates, serum, and other complex biochemical mixtures. The label permits 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 is indicative of a proteolytic event at the sequenced amino acid site. This method is being applied to the study of proteolysis in several different biological systems including cellular programmed cell death, or apoptosis, inflammation, and human serum. This work will shed new light on the biology of apoptosis, inflammation, serum, and the relationship between proteolysis in each of these systems in health and disease. Furthermore, this work will enable continued refinement of a novel proteomic method that we hope 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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的蛋白水解事件的方法受到严重的限制，并且远远落后于通常用于其他翻译后修饰如磷酸化和泛素化的蛋白质组学分析的方法。 我们工作的目的是开发和应用一种新的方法，用于复杂生化混合物中蛋白质水解的全局分析，该方法灵敏、稳健且通用。 该方法基于使用工程化肽连接酶来选择性地标记细胞裂解物、血清和其它复杂生化混合物中的蛋白质N-末端。 该标记允许亲和纯化和富集N-末端肽，用于随后通过串联质谱法进行测序。 鉴定实验样品中存在的N-末端和对照样品中不存在的N-末端指示在测序的氨基酸位点处的蛋白水解事件。 这种方法正被应用于研究蛋白水解在几个不同的生物系统，包括细胞程序性细胞死亡，或凋亡，炎症，和人血清。 这项工作将为细胞凋亡、炎症、血清的生物学以及这些系统中蛋白质水解在健康和疾病中的关系提供新的线索。 此外，这项工作将使一种新的蛋白质组学方法，我们希望将发现应用于生物学的其他领域的蛋白质水解的研究继续完善。 UCSF质谱设施将提供质谱仪器，软件和这项工作的成功所必需的专业知识。