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

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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.