11 Isoelectric focusing and proteomics

Abstract

“Proteomics” can be defined as the ability to conduct high-throughput biochemistry or protein chemistry on a scale comparable with that achieved by molecular biology and its high-throughput counterpart, genomics. All of the original techniques used for the separation of single unique proteins from complex mixtures relied on isoelectric focusing (IEF) as a preliminary step, especially in two-dimensional gel electrophoresis (2-DE) applications. This chapter discusses the proteomics aspect of IEF, focusing on its role in the prefractionation of biological samples and as the preliminary step in 2-DE, with an emphasis on reviewing most recent developments. Currently, two main methods exist for the separation of proteins in proteomics projects: (i) 2-DE and (ii) 2-D liquid chromatography (2-DLC). This chapter concentrates on 2-DE technology. Proteomics is generally applied to questions of biological significance, and the experimental design is aimed at finding biomarkers, irrespective of whether they are the diagnostic of disease, quality in agricultural crops, or pathogenicity factors in infectious disease. IEF is making a significant contribution to prefractionating proteins prior to conventional separation (2-DE and 2-DLC approaches), and to protein identification and characterization (mainly performed using MS). There are two main approaches where IEF has made an impact on prefractionation: (1) the separation of subcellular fractions and organelles and (2) the separation of proteins into pI fractions suitable for use with micro-range immobilized pH gradients (IPG) or 2-DLC-MS, thus aiding in the identification of lower abundance proteins. Each of these approaches is leading to an improved resolution and proteome “coverage” than that can be achieved with currently available conventional technology.