Evolving and Engineering New Protease Tools for Mass Spectrometry Proteomics

描述（由申请人提供） 翻译后摘要：蛋白质翻译后修饰在细胞信号转导和代谢调控中发挥着关键作用。理解生物学的这一基本方面及其在疾病病理学中的意义已经成为蛋白质组学研究的中心目标。为了实现这一目标，质谱法是大规模鉴定蛋白质和翻译后修饰图谱和发现的首选分析方法。然而，蛋白质序列的大部分区域仍然未映射，因为它们缺乏蛋白水解切割位点。拟议的研究计划将通过正交蛋白酶的体外进化来解决这一关键缺点，正交蛋白酶将引入新的切割位点，从而直接在修饰残基附近覆盖序列。将开发一种微流体区室化的体外选择策略，以同时调查1010种突变蛋白酶，并富集表现出新的切割特异性的高活性突变体。富集的突变体池将进行更传统的高通量筛选，以鉴定具有非凡活性、稳健性和特异性的突变体。采用这种策略，新的蛋白酶将被培育成在十种常见的翻译后修饰之一上特异性切割。这些修饰不仅在信号传导和转录调控中是重要的，而且它们的化学独特性将使发现能够识别和切割邻近这些部分的突变蛋白酶的可能性最大化。突变蛋白酶将立即为斯克里普斯佛罗里达管道中的样品产生完整的序列覆盖，包括病毒调节蛋白和癌症相关激酶，它们都在序列覆盖缺口中具有调节修饰。更广泛地说，随着每种新的蛋白酶工具的发展，进行修饰特异性蛋白质数字化的能力将把基于质谱的蛋白质组学从鸟枪淘选技术转变为有针对性的搜索和发现平台。 公共卫生相关性：蛋白质翻译后修饰是代谢的化学开关和旋钮，它们的状态和类型告诉我们细胞功能和调节的分子基础。我们正在利用进化来产生新的分子工具，这些工具将专门揭示这些修饰的存在，将映射技术从随机的淘选探险转变为高度针对性的调查。这些有针对性的映射活动将导致更多的全球性理解修饰如何控制基本的细胞过程，以及病毒和癌症等疾病如何劫持这些控制。

DESCRIPTION (Provided by the applicant) Abstract: Protein post-translational modifications play a key role in cellular signaling and metabolic regulation. Understanding this fundamental aspect of biology and its implications in disease pathology has emerged as a central goal of proteomics research. In pursuit of this goal, mass spectrometry is the analytical method of choice for large-scale identification of proteins and post-translational modification mapping and discovery. However, vast regions of protein sequence remain unmapped because they lack proteolytic cleavage sites. The proposed research program will address this critical shortcoming by in vitro evolution of orthogonal proteases that will introduce new cleavage sites and thereby sequence coverage directly in the vicinity of modified residues. A microfluidic compartmentalized in vitro selection strategy will be developed to survey 1010 mutant proteases simultaneously and enrich in highly active mutants that exhibit novel cleavage specificity. Enriched mutant pools will then be subject to more traditional high-throughput screening to identify mutants with extraordinary activity, robustness, and specificity. Employing this strategy, new proteases will be bred to cleave specifically at one of ten common post-translational modifications. These modifications are not only important in signaling and transcriptional regulation, but their chemical distinctiveness will maximize the probability of discovering mutant proteases capable of recognizing and cleaving adjacent these moieties. The mutant proteases will immediately generate complete sequence coverage for samples in the Scripps Florida pipeline, including viral regulatory proteins and cancer-related kinases that both harbor regulatory modifications in sequence coverage gaps. More broadly, the ability to conduct modification-specific protein digestions will transform mass spectrometry-based proteomics from a shotgun panning technology into a targeted search and discovery platform as each new protease tool is evolved. Public Health Relevance: Protein post-translational modifications are the chemical switches and knobs of metabolism, and their state and type teach us the molecular basis of cellular function and regulation. We are using evolution to generate new molecular tools that will specifically reveal the presence of these modifications, transforming mapping technology from a random panning expedition into a highly targeted survey. These targeted mapping campaigns will lead to more global understanding of how modifications control basic cellular processes and how diseases such as viruses and cancers hijack these controls.