Redox proteomics in the blue mussel Mytilus edulis: Carbonylation is not a pre-requisite for ubiquitination in acute free radical-mediated oxidative stress

Abstract

Mytilus edulis was exposed under controlled conditions to a panel of model pro-oxidants (H 2O 2, CdCl 2 and menadione) for 24 h. Protein extracts of gill, mantle and digestive gland were analysed by immunoblotting in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separations. Immunoblotting revealed extensive and comparable levels of protein carbonylation across the pro-oxidant panel with approximately 1.5-fold higher levels in gill than digestive gland. Ubiquitination in gill was modest in response to H 2O 2, but increased in response to menadione and CdCl 2. High ubiquitination levels were found for all pro-oxidants in digestive gland with levels comparable to the highest found in gill. Two-dimensional (2D) SDS-PAGE confirmed specific targeting of individual proteins by ubiquitin against a generally stable protein expression signature. Spot matching suggested that carbonylation is not a pre-requisite for ubiquitination. While gill showed consistently higher constitutive levels of glutathione transferase, glucose 6-phosphate dehydrogenase and glutathione reductase activity, pro-oxidant treatment had only modest effect on these enzymes and on the ratio of reduced/oxidised glutathione. In digestive gland, this latter ratio was higher than in gill and increased in response to menadione and CdCl 2. Ubiquitination may provide a marker for acute onset of oxidative stress in bivalves.