Prefractionation of proteins based on electrokinetic methodologies in free solution essentially relaying on the isoeletric focusing (IEF) has gained wide acceptance. Many commercial devices are now constructed to take the advantage of this principle ( Table 2 ). Reproducible fractionation steps will break down the sample complexicity while concentrating low abundant species, resulting in more confident protein identifications and quantification by 2D gels, mass spectrometry, and protein arrays. A good example of a innovation is liquid-phase isoelectric focusing (IEF) as a prefractionation tool before the first dimension of 2D gel electrophoresis [118, 119] . For more consistent pI separation, the Zoom IEF fractionator [120] and multicompartment electrolyser (MCE) [121] are being used to prefractionate the proteins. The fractionated samples can be directly applied on standard narrow range IPG strips for 2D electrophoresis. This allows at least 10000 to 15000 separate proteins to be analyzed, including proteins of very low abundance. IEF, a highresolution electrophoresis technique, has been widely used in shotgun proteomic experiments [122] . IEF runs in a buffer-free solution containing carrier ampholytes or in immobilized pH gradient (IPG) gels. The use of IPG-IEF for the separation of complex peptide mixtures has been applied to the analysis of plasma and amniotic fluid [123, 124] as well as to bacterial material [125] . The IPG gel strip is divided into small sections for extraction and cleaning up of the peptides. This technique recovers the sample from the liquid phase and was demonstrated to be of great interest in shotgun proteomics [126] . IEF is not only a high resolution and high capacity separation method for peptides, it also provides additional physicochemical information like their isoelectric point [127, 128] . The pI value provided is used as an independent validating and filtering tool during database search for MS/MS peptide sequence identification [129] . The recent introduction of commercially available OFFGEL fractionator system by Agilent Technologies provides an efficient and reproducible separation technique [130] . This separation is based on immobilized pH gradient (IPG) strips and permits to separate peptides and proteins according to their isoelectric point (pI) but is realized in solution [131] . Its micropreparative scale provides fraction volumes large enough to perform subsequent analyses as reverse phase (RP)-liquid chromatography (LC)-MALDI MS/MS. The combined use of iTRAQ labeling and OFFGEL fractionation methods for the proteomic study of complex sample is also being considered [132, 133] .