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Mitochondria are often referred to as the cellular powerhouse due to their key function in the process of oxidative phosphorylation. But side reactions of the electron transport chain are implemented in the generation of reactive oxygen species. These ROS are highly reactive and cause damage to biological molecules. The free radical theory of ageing hypothesizes that such damage is the primary mechanism of ageing. Therefore the search for proteins that are targets of damage is of critical importance to understand the role of oxidative stress in ageing. Proteomics combined with MS provides a powerful suite of techniques for the identification of these proteins. We focused on the development and refinement of a proteomic workflow aiming to produce a high quality quantitative survey of the mitochondrial proteome. We performed qualitative LC-MS/MS-based experiments on mitochondria proteins by means of both gel-based and gel-free approaches, showing an over 4-fold increase in protein identifications by the latter. In addition first quantitative results were collected, yielding in the quantitation of 599 proteins, illustrating the potential of the developed method and the acquired data.