Joseph Robert Burgoyne1, Olujimi Oviosu, Philip Eaton. 1. King's College London, Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK. Electronic address: joseph.burgoyne@kcl.ac.uk.
Abstract
INTRODUCTION: Reversible protein cysteine oxidation is recognised as a pivotal post-transitional modification that transduces physiological as well as pathological signalling. Pharmacological interventions that target specific sources of oxidant formation are currently being trialled to ascertain their potential ability to prevent disease progression. To determine the selectivity of such pharmacological treatments and to indentify new drug targets, a suitable method is required to detect target cysteine oxidation. METHOD: Using a polyethylene glycol (PEG)-based alkylating agent the reversible oxidation of target proteins can be determined using a novel switch method. After reduction and specific labelling of reversibly oxidised thiols with a 'heavy' PEG-tag, samples are resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, Western blotted and immunostained for protein(s) of interest. A mobility shift in a target protein following PEG-alkylation correlates with the reversible oxidative modification. RESULTS: The oxidation of cAMP- and cGMP-dependent protein kinases was detected using the PEG-switch assay in Langendorff-perfused hearts after hydrogen peroxide was administered. DISCUSSION: The PEG-switch assay is a fast effective semi-quantitative method for measuring target reversible cysteine oxidation in complex protein mixtures derived from tissue or cultured cells.
INTRODUCTION: Reversible protein cysteine oxidation is recognised as a pivotal post-transitional modification that transduces physiological as well as pathological signalling. Pharmacological interventions that target specific sources of oxidant formation are currently being trialled to ascertain their potential ability to prevent disease progression. To determine the selectivity of such pharmacological treatments and to indentify new drug targets, a suitable method is required to detect target cysteine oxidation. METHOD: Using a polyethylene glycol (PEG)-based alkylating agent the reversible oxidation of target proteins can be determined using a novel switch method. After reduction and specific labelling of reversibly oxidised thiols with a 'heavy' PEG-tag, samples are resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, Western blotted and immunostained for protein(s) of interest. A mobility shift in a target protein following PEG-alkylation correlates with the reversible oxidative modification. RESULTS: The oxidation of cAMP- and cGMP-dependent protein kinases was detected using the PEG-switch assay in Langendorff-perfused hearts after hydrogen peroxide was administered. DISCUSSION: The PEG-switch assay is a fast effective semi-quantitative method for measuring target reversible cysteine oxidation in complex protein mixtures derived from tissue or cultured cells.
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