Secretion of outer membrane vesicles (OMV) presents an important phenomenon in Gram-negative bacteria and they play multiple roles in their lifestyle including virulence and host-pathogen interaction. Francisella tularensis secretes unusually shaped tubular OMV. We here present a proteomic comparison of whole cell lysates, OMV and membrane fractions derived from two F. tularensis strains: a moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis SchuS4 strain. We describe a novel approach to the cross-species proteomic comparison based on creating an intersection protein database from the individual single-species databases. This approach proved to be less prone to quantification errors arising from differences in the protein sequences than using the individual databases. The consecutive comparison of the subproteomes of OMV and membranes of the two strains allowed us to distinguish the differences in protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membrane. Amongst the most different proteins we detected proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some of the major structural outer membrane proteins.