Search

Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method.

The study of the “proteomes” of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.

Today the omnipresence of MS-based proteomic approaches suggest, that 2-dimensional gel (2-DE) based proteomics is dead. In reality, this is far from the truth, and also after three decades high-resolution 2-DE, introduced by Patrick O’Farrell in 1975, still represents a key technology to investigate thousands of protein species, their isoforms and modifications simultaneously. Especially, in combination with multi fluorescence labelling and various MS-methodologies, it is very popular to relatively quantify and identify proteins in complex mixtures. But also after these 30 years of use, reproducibility and process stability are still under constant improvement to further enhance robustness and feasibility of the method. In order to offer an opportunity to discuss and apply the latest developments in 2-DE in a scientific context we picked out this technology as central theme for the International Protein Rainbow Workshop titled ‘‘2-Dimensional Gel Electrophoresis Reloaded’’, which was held at the Institute of Clinical Biochemistry and Pathobiochemistry at the German Diabetes-Center in Duesseldorf. This annual hands-on training course, comprising theoretical and practical aspects of the technology, was accompanied by a lecture day giving an overview of recent 2-DE applications in scientific research. In dependence on this the current issue ‘‘2-Dimensional Gel Electrophoresis Reloaded’’ should help to give an overview of recent developments and applications related to 2-DE. The collection of scientific reports covers a broad range of applications, starting with an inter-laboratory comparison introducing a simplified and robust 2-DE work-flow. In addition a novel fluorescent dye for improved protein detection is described and pre-analytical conditions for serum proteomics, representing one crucial point for biomarker discovery are addressed. Another study examines differences and similarities between protein patterns derived from mitochondria and peroxisomes or describe the characterization of sub-types in small cell lung cancer (SCLC).