# Developing chemical-proteomic tools to investigate cysteine oxidation

> **NIH NIH R35** · BOSTON COLLEGE · 2020 · $349,518

## Abstract

Project Summary
 Various cellular processes generate reactive oxygen species (ROS) such as superoxide
and hydrogen peroxide, which are key mediators of signaling pathways. Dysregulated redox
homeostasis and ROS generation are hallmarks of diseases including cancer and age-
associated degeneration. Primary cellular targets of ROS are cysteine thiols that form sulfenic,
sulfinic, and sulfonic acids, as well as disulfides. ROS generation is localized to specific cellular
regions, and are short-lived, necessitating spatiotemporal methods to study cysteine oxidation
events. We have developed a chemical-proteomic strategy, termed isoTOP-ABPP, to study
diverse oxidative cysteine modifications. To provide the necessary spatiotemporal readout, we
will expand our current isoTOP-ABB platform by: (1) utilizing caged cysteine-reactive probes
that can be activated rapidly by light for temporal control of cysteine labeling; and, (2) adapting
the TurboID proximity biotinylation method to spatially localize our analysis to specific regions of
the cell. We will apply these spatiotemporal readouts to two ROS-generating systems in the cell:
(1) growth-factor mediated activation of NADPH oxidase (NOX); and, (2) inhibition of the
electron transport chain (ETC) and mitochondrial superoxide dismutase.
 Epidermal growth factor (EGF) binding to EGFR, activates ROS release via NOX2. We
aim to identify cysteine oxidation events proximal to NOX2 and characterize the role of these
oxidation events on growth-factor signaling. In early studies, we identified a redox-active
disulfide bond in a fatty-acid binding protein, FABP5, which is formed upon EGF stimulation of
A431 cells. We will investigate the effects of disulfide-bond formation on the lipid-binding
properties of FABP5, and downstream signaling pathways mediated by growth-factor
stimulation.
 The mitochondria are highly redox-active organelles, and we aim to provide a
comprehensive view of oxidation targets during mitochondrial dysfunction, by selectively
enriching mitochondrial proteins prior to analysis. We will apply our optimized mitochondrial
methods to interrogate cysteine oxidation in a C. elegans SOD-2 mutant with elevated
mitochondrial ROS, and extended lifespan.

## Key facts

- **NIH application ID:** 9851530
- **Project number:** 1R35GM134964-01
- **Recipient organization:** BOSTON COLLEGE
- **Principal Investigator:** Eranthie Weerapana
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $349,518
- **Award type:** 1
- **Project period:** 2020-01-01 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9851530

## Citation

> US National Institutes of Health, RePORTER application 9851530, Developing chemical-proteomic tools to investigate cysteine oxidation (1R35GM134964-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9851530. Licensed CC0.

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