Updated project metadata. Lyme borreliosis is first characterized by a cutaneous inflammation, the erythema migrans, and if dissemination occurs after an infected tick bite cutaneous, nervous and articular manifestations appear. Although antibiotic treatments are efficient in the early stage of the infection, a significant number of patients develop disseminated manifestations due to unnoticed or absence of erythema migrans, or to inappropriate treatment. Vaccine could be an efficient approach to decrease Lyme disease incidence. We have developed a proteomic approach based on a Ge-LC-MS/MS strategy to identify new vaccine candidates. We analyzed a disseminating clone and the associated wild type strain for each major pathogenic Borrelia species: B. burgdorferi sensu stricto, B. garinii and B. afzelii. We managed to identify proteins specific and common to the disseminating clones of the 3 main species. In parallel, we used a spectral counting strategy in order to identify up-regulated proteins common to the clones. Thus, we identified 40 proteins that are potentially involved in bacterial virulence and could be of interest in the development of a new vaccine.