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

> **NIH NIH R01** · UNIVERSITY OF MARYLAND BALTIMORE · 2021 · $263,853

## Abstract

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.

## Key facts

- **NIH application ID:** 10125167
- **Project number:** 5R01GM127595-04
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Lisa M Jones
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $263,853
- **Award type:** 5
- **Project period:** 2018-06-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10125167

## Citation

> US National Institutes of Health, RePORTER application 10125167, Development of an In Vivo Footprinting Method Coupled with Mass Spectrometry in C. elegans (5R01GM127595-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10125167. Licensed CC0.

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