INTRODUCTION The molecular scanner offers a flexible and powerful visualization tool that can create a fully annotated 2D gel electrophoresis map. Proteins separated by 2D gel electrophoresis are simultaneously digested while undergoing electrotransfer from the gel to a membrane. The peptides are subjected to peptide mass fingerprint (PMF) analysis to identify proteins directly from the PVDF membranes by MALDI-TOF-MS scanning. An ensemble of dedicated tools is then used to create, analyze, and visualize a proteome as a multidimensional image. The molecular scanner method reduces to a minimum the sample handling prior to mass analysis and decreases the sample size to a few tens of micrometers, that is, the size of the MALDI-TOF-MS laser beam impact. The process can be divided into four parts: separation and digestion of proteins, acquisition of PMF data, processing of the MS data and protein identification, and creation of multidimensional proteome maps.