Evolving and Engineering New Protease Tools for Mass Spectrometry Proteomics

描述(由申请人提供) 摘要：蛋白质翻译后修饰在细胞信号和代谢调节中起着关键作用。了解生物学的这一基本方面及其在疾病病理学中的意义已成为蛋白质组学研究的中心目标。为了追求这一目标，质谱学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没有被定位，因为它们缺乏蛋白水解性裂解位点。拟议的研究计划将通过在体外进化正交蛋白酶来解决这一关键缺陷，该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。将开发一种微流控分室体外选择策略，同时检测1010个突变的蛋白水解酶，并丰富表现出新的切割特异性的高活性突变。然后，丰富的突变体库将接受更传统的高通量筛选，以确定具有非凡活性、稳健性和特异性的突变。采用这一策略，新的蛋白水解酶将被培育成在十种常见的翻译后修饰中的一种特定切割。这些修饰不仅在信号和转录调控方面很重要，而且它们的化学独特性将最大限度地增加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。突变的蛋白酶将立即为佛罗里达州斯克里普斯管道中的样本生成完整的序列覆盖，包括病毒调节蛋白和癌症相关蛋白，它们都在序列覆盖缺口中包含调节修饰。更广泛地说，随着每一种新的蛋白酶工具的发展，进行特定修饰的蛋白质消化的能力将把基于质谱学的蛋白质组学从鸟枪式淘洗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新陈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。我们正在利用进化论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，将测绘技术从随机的淘金探险转变为高度有针对性的调查。这些有针对性的测绘活动将使全球更多地了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.