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Published on: June 21, 2021
The PEG-switch assay: a fast semi-quantitative method to determine protein reversible cysteine oxidation
Joseph Robert Burgoyne1, Olujimi Oviosu, Philip Eaton
1King's College London, Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK.
Journal of Pharmacological and Toxicological Methods
|July 17, 2013
Summary
A new PEG-switch assay detects reversible protein cysteine oxidation, a key signaling modification. This method aids in developing targeted therapies for diseases involving oxidative stress.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Signaling
Background:
- Reversible protein cysteine oxidation is a critical post-translational modification involved in physiological and pathological signaling pathways.
- Pharmacological strategies targeting oxidant formation are under investigation for disease prevention, necessitating methods to assess drug selectivity.
Purpose of the Study:
- To develop and validate a novel method for detecting and quantifying target protein cysteine oxidation.
- To enable the identification of new drug targets and assess the selectivity of pharmacological interventions.
Main Methods:
- A polyethylene glycol (PEG)-based alkylating agent and a novel 'switch' method were employed.
- Reversibly oxidized thiols were labeled with a 'heavy' PEG-tag after reduction.
- Samples were analyzed using SDS-PAGE, Western blotting, and immunostaining, with a mobility shift indicating oxidation.
Main Results:
- The PEG-switch assay successfully detected the oxidation of cAMP- and cGMP-dependent protein kinases.
- Oxidation was observed in Langendorff-perfused hearts following hydrogen peroxide administration.
Conclusions:
- The PEG-switch assay provides a rapid, effective, and semi-quantitative method for measuring reversible cysteine oxidation.
- This assay is applicable to complex protein mixtures from various biological sources, including tissues and cultured cells.
