Two-Dimensional Gel Electrophoresis (2-DE)

Two-dimensional gel electrophoresis (2-DE) is able to separate hundreds to thousands of proteins or polypeptides by coupling IsoElectric Focusing (IEF) in first dimension and Sodium Dodecyl Sulphate PolyAcrylamide-Gel Electrophoresis (SDS-PAGE) in second dimension. This particular configuration is called classical 2-DE: IEF separates proteins in function of their isoelectric point (pI) and SDS-PAGE in function of their molecular mass (Mr), these two parameters being unrelated; the second dimension can be also Native PAGE. In other configurations, Native PAGE is the first dimension and SDS-PAGE the second one. Classical 2-DE is still the core technique in proteomics as the first step to separate complex protein mixtures, the second step being the identification of these separated polypeptides using mass spectrometry, either with peptide mass fingerprinting (PMF) after specific proteolytic hydrolysis (for MALDI-TOF or ESI-TOF mass spectrometry), or with sequencing of the polypeptide chain using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Classical 2-DE has also direct applications, such as phenotyping of genetic variants and post-translational modification (PTM) characterization, in particular phosphorylations, glycosylations, deamidations and much more. This chapter will describe the main 2-DE techniques with some developments on IEF, Native PAGE as first or second dimension, and SDS-PAGE as second dimension in proteomic analysis.