Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples.It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques:isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension).2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell:its proteome.Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmentsl and medically-relevant bacteria.Here we give a broad overview of the basic principles and historical development of gel-based proteomics,and how this powerful approach can be applied for studying bacterial biology and physiology.We highlight specific 2-DE applications that can be used to analyze when,where and how much proteins are expressed.The links between proteomics,genomics and mass spectrometry are discussed.We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing.The use of proteome fractionation techniques and non-gel-based proteemic approaches are also discussed.We highlight how the analysis of protsins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response.This review is aimed at non-specialists who wish to gain a concise,comprehensive and contemporary overview of the nature and applications of bacterial proteomics.