Currently no single proteomic technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the PF2D system from Beckman Coulter, 1-D reverse-phase high performance liquid chromatography and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped, with less than 24% of proteins common between any two technologies, and only 7% common amongst all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. Eighty two percent of these proteins had not been previously observed in proteomic studies of this subproteome, whereas 44% had not been identified in proteomic studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. As well, we demonstrated that both the 1- and 2-DLC allowed for the separation of the a-subunit of F1Fo ATP synthase which differed due to a change in pI or hydrophobicity.