Proteomics is required to provide insight into any type of subproteome. While the workflow based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) can be applied for many subproteomes and comprises well-established strategies for data presentation and data analysis, the comprehensive investigation of membrane proteomes remains a challenging task. We present a number of procedures that provide an insight into such systems. We have established a novel protocol for the efficient preparation of membrane fractions, which is used here for the human pathogen Listeria monocytogenes that overcomes difficulties associated with ribosomes. Subsequently, we have used the combination of sodium dodecyl sulfate (SDS)-PAGE and liquid chromatography - tandem mass spectrometry for the characterization of the membrane proteome. Three hundred and one different membrane proteins could be identified, including 70 proteins that exhibited 2-15 transmembrane domains. However, a remarkably high ratio of proteins was detected in gel sections that were not in accordance with their expected migration behavior during SDS-PAGE. Protein identifications based on MASCOT significance criteria could be shown to be of high quality and therefore could not be the explanation of this observation. Consequently we have developed LaneSpector, a general visualization tool that allows the systematic comparison between apparent and calculated protein masses, which is routinely applicable to any high-throughput approach using a mass-dependent separation dimension prior to LC-MS/MS. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and might help to reveal relevant biological processes such as proteolysis or other post-translational modifications.