Beside bioreactor modeling studies, the molecular characterization of life-support organisms appeared to be essential to complete their global behavior picture, in particular, culture conditions. Using a combination of LC-MS/MS approaches with gel-free and gel-based peptides fractionation steps, we identified 932 proteins from the alpha-proteobacterium Rhodospirillum rubrum S1H. In addition, abundance data were retrieved using the recently developed emPAI approach which takes into account the number of sequenced peptides per protein. This work has also allowed identification of new and specific proteins for the Rhodospirillaceae family.