Development of an In Vivo Footprinting Method Coupled with Mass Spectrometry in C. elegans

线虫体内足迹法与质谱联用的发展

• 批准号: 9904716
• 负责人: Lisa M Jones
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904716
• 关键词: Address; Animal Model; Binding Sites; Biological Models; Blood capillaries; Caenorhabditis elegans; Calcium; Calcium-Binding Proteins; Caliber; Calmodulin; Calmodulin-Binding Proteins; Cell Culture Techniques; Cells; Chemicals; Chromatography; Coupled; Crystallization; Cytolysis; Data; Detection; Development; Dimethyl Sulfoxide; Disease; Enhancers; Ensure; Fractionation; Goals; Grant; Hydrogen Peroxide; Hydroxyl Radical; In Vitro; Label; Lasers; Lead; Life Cycle Stages; Ligand Binding; Ligands; Light; Mass Spectrum Analysis; Methods; Modeling; Modification; Molecular Conformation; Pathogenesis; Pattern; Penetration; Peptides; Peroxides; Protein Footprinting; Proteins; Resolution; Role; Sampling; Site; Solvents; Structure; System; Tail; Testing; Time; Tube; base; biological systems; exoskeleton; experimental study; human disease; in vivo; in vivo evaluation; novel; oxidation; photolysis; protein structure; structural biology; tool; two-dimensional; uptake

PROJECT SUMMARY Studying protein structure in vivo, in an animal model, will provide a wealth of information on the role of protein structure in human disease. Animal models provide a more detailed view of disease pathogenesis owing to presence of interacting systems. Currently available in vivo structural methods are limited in the resolution of information they can provide making the development of new methods essential. Our long-term goal is to study the role of protein structure in the pathogenesis of human disease, in particular, using C. elegans as an animal model for many different diseases. The objective of this grant is to develop a mass spectrometry-based method to analyze protein structure in vivo. This method, entitled in vivo fast photochemical oxidation of proteins (IV-FPOP), utilizes hydroxyl radicals to oxidatively modify solvent accessible sites in proteins. As solvent accessibility changes upon ligand binding or a conformational change, a differential experiment such as ligand bound vs. ligand free can identify protein interactions sites and regions of conformational change. The efficacy of the method depends on hydrogen peroxide uptake by the worm, the ability of the worms to flow through a flow tube, and mass spectrometry detection. We will address these issues by optimizing the use the of chemical penetration enhancers and hydrogen peroxide concentration to obtain the highest amount of peroxide uptake in the shortest amount of time (specific aim 1). To ensure single worm flow, we will optimize the size of the flow capillary (specific aim 2). To increase the identification of peptides by mass spectrometry, we will optimize worm lysis, two-dimensional chromatography, and sample fractionation (specific aim 3). We will also analyze the protein calmodulin as a model protein for determining whether the method can identify protein interaction sites and regions of conformational change in vivo (specific aim 3). Worms will be oxidatively modified in the presence and absence of calcium and the differences in modification pattern for calmodulin will be examined. The developed method would provide a new tool for the structural biology toolbox that has several advantages over currently available in vivo methods.

项目摘要 在动物模型中研究体内蛋白质结构将提供有关该作用的大量信息 人类疾病中蛋白质结构的结构。动物模型提供了更详细的疾病观点 由于存在相互作用系统，发病机理。目前可用于体内结构方法 限制了他们可以提供的信息解决新方法的开发 基本的。我们的长期目标是研究蛋白质结构在人体发病机理中的作用 尤其是使用秀丽隐杆线虫作为许多不同疾病的动物模型。目的 该赠款是为了开发一种基于质谱的方法来分析体内蛋白质结构。这 方法为蛋白质（IV-FPOP）的体内快速光化学氧化，利用羟基自由基 以氧化修饰蛋白质中的溶剂可访问位点。随着配体上的溶剂可及性的变化 结合或构象变化，差异实验，例如配体结合与配体的无配体可以 识别蛋白质相互作用位点和构象变化的区域。该方法的功效 取决于蠕虫的过氧化氢摄取，蠕虫流动流动的能力 管和质谱检测。我们将通过优化使用的使用来解决这些问题 化学渗透增强剂和过氧​​化氢浓度以获得最高量 在最短的时间内过氧化物的吸收（特定目标1）。为了确保单蠕虫流动，我们将 优化流毛细管的大小（特定目标2）。增加肽对肽的识别 质谱法，我们将优化蠕虫裂解，二维色谱和样品 分馏（特定目标3）。我们还将分析蛋白质钙调蛋白作为模型蛋白 确定该方法是否可以识别构象的蛋白质相互作用位点和区域 变化体内（特定目标3）。在存在和不存在的情况下，蠕虫将被氧化修饰 钙和钙调蛋白的修饰模式的差异将被检查。发达的 方法将为结构生物学工具箱提供一个新的工具，该工具箱具有多个优势 目前可用于体内方法。