More than 30 years ago people have learned that exercise promotes oxidative damage in human tissues. Since then, our knowledge on exercise-induced free radical production and their effect has advanced markedly. In the beginning research has mainly focused on detrimental effects of exercise-induced free radical production (e.g. oxidation of macromolecules); however today a new era of redox biology exist that centers on the cell-signaling effects of exercise-induced free radicals, such as reactive oxygen species (ROS) that were shown to play a crucial role in muscle adaptation to exercise. Erythrocytes are rich in membrane polyunsaturated free fatty acid content and have high cellular concentrations of oxygen and haemoglobin that makes them susceptible to ROS induced damage. However, mild oxidative stress has beneficial effect by increasing endogenous antioxidant mechanisms. Thus, in the present work we have used sensitive mass spectrometry techniques to assess the potentially beneficial oxidative modifications of erythrocyte membrane proteins in response to exercise. We have recruited 3 healthy volunteers and their blood was drawn before exercise, immediately after and few hours after exercise. Erythrocyte membranes ("ghosts") were prepared and membrane proteins isolated. Proteins were then separated by 1-D gel electrophoresis, digested with trypsin and peptides analyzed by Orbitrap ELITE coupled with the Easy n-LC II for nano LC-MS/MS. The acquired data was searched with Proteome Discoverer 1.4 against Homo sapiens database to reveal the changes in the erythrocyte membrane proteome. Furthermore, data acquired was also searched through the database using the comprehensive workflow that included several dynamic modifications of peptides, including N-terminal carboxymethyl, C-terminal oxidation and terminal independent oxidation and trioxidation of peptides. The results obtained revealed several proteins that have translocated from the cytoplasm into the membrane following exercise. Moreover, numerous proteins showed exercise dependent modifications and up/down regulation. Exercise induced proteome changes were demonstrated to play an important role in several signaling pathways related to healthy living and lifestyle.


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