The influence of cigarette smoke free radicals on cell lines has been investigated in vitro by many methods: Ames tests for detecting mutagenesis; DNA adducts formation and more recently gene expression using RNA arrays. To the best of our knowledge, proteomics has not been used for investigating the influence of cigarette smoke components. Indeed, proteomics studies are very complementary to transcriptomics studies, as they give access to the modification of protein expression in cells under various conditions. Our first investigations were performed on MCF-7 cells that are well known in our laboratory. Untreated cells, cells incubated with conventional cigarette smoke solution and with free radical depleted cigarette smoke solution were compared. We used difference in-gel electrophoresis (DIGE) based on labelling proteins from treated and untreated cells by fluoresced reagent Cy3 and Cy5 and co-migrating the proteins in order to identify the differentially expressed proteins. The image analysis revealed that several hundred proteins are differentially expressed and several tenth changes in post-translational modification profiles. The more dramatic change is observed between cells treated with native and free radicals depleted cigarette smoke solution. We then identified the more intense differentially expressed proteins using peptide mass fingerprinting. Briefly, spots are excised, proteins are digested in gel, the peptide eluted and analyzed by MALDI-TOF mass spectrometry. The proteins are then identified by comparing in silico digestion of proteins contained in data bank and the experimental spectrum using a search engine, in our case Mascot. All the identified proteins behave to the oxidative stress pathway. Nano-LC-nano-ESI MS/MS on our 9.4 Tesla FT-MS instrument which allows to separate each individual peptide and to sequence it was further used to confirm those identifications and to investigate the post-translational modifications. Dose-response relationships and use of more relevant cell lines are currently under investigation in our laboratory.