Sample Preparation and Prefractionation Techniques for Electrophoresis-Based Proteomics

In the 1990s much of the technology development for 2-D gels was centred on attempts to solubilise and separate whole proteomes. In the last 10 years, much has changed in the science of sample preparation, but the art of 2-D gels is almost the same, and remains a method that many love to hate. The improvements in protein solubilisation, the introduction of pH gradients that extend into the far alkaline region, and particularly the hunt for lowabundance proteins have served to hasten the arrival of the most important current area of sample preparation – fractionation. As proteomics matures, there is a new focus on function and the analysis of co- and post-translational modifications. With this in mind, and as the complexity of sample preparations increases, this chapter deals with possible artefacts and how to avoid generating them. Finally, with artefacts banished, this chapter reviews the most appealing prefractionation tools that are currently available. Fractionation is discussed as a formidable tool for mining below the tip of the proteomic iceberg.