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

项目摘要 在动物模型中研究体内蛋白质结构，将提供大量关于蛋白质结构的作用的信息。 蛋白质结构的变化。动物模型提供了更详细的疾病视图 发病机制由于相互作用系统的存在。目前可用的体内结构方法是 它们所能提供的信息分辨率有限， 具有本质意义我们的长期目标是研究蛋白质结构在人类肿瘤发病机制中的作用， 疾病，特别是使用C.线虫作为许多不同疾病的动物模型。的目标 这项拨款是为了发展一种以质谱为基础的方法来分析体内蛋白质结构。这 一种名为体内蛋白质快速光化学氧化（IV-FPOP）的方法，利用羟基自由基 以氧化方式修饰蛋白质中的溶剂可及位点。由于溶剂可及性随配体变化 结合或构象变化，差异实验如配体结合与配体游离可 确定蛋白质相互作用位点和构象变化区域。该方法的有效性 取决于蠕虫对过氧化氢的吸收，蠕虫流过水流的能力， 管和质谱检测。我们将通过优化使用 化学渗透促进剂和过氧化氢浓度，以获得最高量的 在最短的时间内吸收过氧化物（具体目标1）。为了确保单蠕虫流量，我们将 优化流动毛细管的尺寸（具体目标2）。通过以下方法增加肽的鉴定 质谱，我们将优化蠕虫裂解，二维色谱，和样品 分离（具体目标3）。我们还将分析蛋白质钙调素作为模型蛋白质， 确定所述方法是否可以鉴定蛋白质相互作用位点和构象可变区， 体内变化（具体目标3）。蠕虫将在存在和不存在以下物质的情况下被氧化修饰： 将检查钙和钙调蛋白修饰模式的差异。发达 方法将为结构生物学工具箱提供一种新的工具， 目前可用的体内方法。

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.