Engineered proteases for proteomics

描述（由申请人提供）：蛋白质组学是一个迅速发展的领域，但目前的方法仍然不足以充分发挥其潜力。表征蛋白质的最基本的酶学工具是蛋白酶。蛋白酶已经是蛋白质组学分析的重要组成部分，但需要更复杂的工具来识别高度复杂样品中的低丰度蛋白质。我们已经设计出在变性条件下具有活性的“限制性内切”蛋白酶原型，并在定义明确的同源序列基序上进行特异性切割。我们的基本创新将是将蛋白质组解析成序列编辑切片的能力，并以高分辨率和高灵敏度检测这些编辑部分。在第一阶段，我们测试了原型限制性内切蛋白酶在蛋白质组学分析中的适用性，并实施了一种新的定向进化方法来选择切割新序列基序的蛋白酶。第二阶段的目标是开发一套复杂的蛋白酶工具，以促进蛋白质组学分析，就像限制性内切酶促进基因组分析一样。

DESCRIPTION (provided by applicant): Proteomics is a rapidly expanding field but current methodologies remain inadequate for achieving its full potential. The most basic enzymological tool for characterizing proteins is the protease. Proteases are already an essential part of proteomic analysis but more sophisticated tools are needed to identify low abundance proteins in highly complex samples. We have engineered prototype "restriction" proteases which are active in denaturing conditions and which cut specifically at well-defined cognate sequence motifs. Our basic innovation would be the ability to parse a proteome into sequence-edited slices and to detect these edited portions with high resolution and high sensitivity. In Phase I we tested the prototype restriction protease for suitability in proteomic analysis and implemented a novel directed evolution methodology for the selection of proteases that cut new sequence motifs. The Phase II objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. PUBLIC HEALTH RELEVANCE: The complex and dynamic nature of proteomes make them rich with useful information but difficult to characterize. Our long-range objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. Better proteomic tools will lead to earlier detection of disease states, better treatments, better predictability of the effects of various treatments, and the development of individualized therapies.