The recent trend in science is to assay as many biological molecules as possible within a single experiment. This trend is evident in proteomics where the aim is to characterize thousands of proteins within cells, tissues, and organisms. While advances in mass spectrometry have been critical, developments made in two-dimensional PAGE (2D-PAGE) have also played a major role in enabling proteomics. In this review, we discuss and highlight the advances made in 2D-PAGE over the past 25 years that have made it a foundational tool in proteomic research.
The last 25 years, and particularly the last decade, has witnessed an increased effort to develop technologies capable of identifying and quantifying large numbers of proteins expressed within a cell system (i.e., the proteome) in the hope of detecting disease biomarkers, mapping protein circuitry, or identifying novel phosphorylation sites, for example. The complexity of the proteome has made developing methods for efficient separation and sensitive detection of proteins a critical component of this effort. Continued advances in mass spectrometry (MS) technology have enabled the detection of proteins with much greater speed and sensitivity than previously possible. Even cutting-edge MS, however, is unable to characterize all of the components within a complex proteome. Scientists take a “divide and conquer” approach to characterizing proteomes, in that they attempt to temporally limit the number of proteins that the mass spectrometer is asked to analyze. By spreading out the proteome, more proteins will ultimately be analyzed within an individual experiment.
To separate proteomes, scientists have used electrophoretic and chromatographic technologies, separately and in combination, and both offline and online. Although these efforts can result in the separation and identification of thousands of proteins, no single method can resolve all the proteins in a proteome, due to their large number and concentration dynamic range. Single-dimension separations are inadequate for effectively resolving complex protein mixtures. This fact was acknowledged over half a century ago by Smithies and Poulik (1), who recognized that a combination of two electrophoretic processes on a gel at right angles should give a much greater degree of resolution than is possible with either separately. The two electrophoretic processes are resolution by molecular size and free solution mobility on a starch gel. Their prediction continues to be proven true and has formed the basis for developing orthogonal multidimensional methodologies for the separation of complex mixtures not only by gel electrophoresis but also by chromatography and capillary electrophoresis.
To properly understand the advances made in two-dimensional PAGE (2D-PAGE), one needs to go back much further than a quarter of a century. In 1930 Tiselius introduced the moving boundary method as an analytical tool for studying the electrophoresis of proteins (2). Since his pioneering work, various forms of electrophoresis have been used for the separation of complex mixtures of proteins, each with improved resolution. As early as 1962, Raymond and Aurell (3) demonstrated the significant nonlinear effects of gel concentration on the electrophoretic mobility of proteins by employing 2-D electrophoresis using different acrylamide gel concentrations to separate serum proteins. Two years later, Raymond (4) demonstrated the superiority of flat slab gels compared with cylindrical tube gels. For example, the flat slab provides maximum surface area for cooling the gel; the resulting patterns are easier to quantify in standard recording densitometers; a large number of samples can be processed using a single gel plate, facilitating the direct comparison of specimens processed under identical conditions; and, most importantly, the flat slab permits the application of 2-D separations. These insightful preferences have been proven true and are practiced today in many bioanalytical laboratories.
Another advancement in 2-D gel separations was introduced in 1972 by Wright (5), who used a 4.75% (2% cross-linkage) polyacrylamide gel column in the first dimension, which was then removed from the glass cylinder and laid on the upper edge of a 2% gradient slab. Following electrophoresis, the gel slab was placed in a staining solution, resulting in the visualization of 112 resolved human serum proteins.
These novel approaches resolved only a small number of proteins, primarily the most abundant proteins of a cell or serum proteome. The introduction of 2D-PAGE in 1975 by O'Farrell (6) for separating cellular proteins under denaturing conditions enabled the resolution of hundreds of proteins. The principle applied was very simple: proteins were resolved on a gel using isoelectric focusing (IEF), which separates proteins in the first dimension according to their isoelectric point, followed by electrophoresis in a second dimension in the presence of sodium dodecyl sulfate (SDS), which separates proteins according to their molecular mass. O'Farrell's method is truly the basis of modern 2D-PAGE, which was quickly adapted and widely accepted by other researchers. Anderson and Anderson (7) used 2D-PAGE for the analysis of human plasma proteins. They were able to separate and detect approximately 300 distinct protein spots upon staining. Unlike O'Farrell, Manabe (8) separated human plasma proteins using 2D-PAGE without denaturing agents. About 230 protein spots could be observed on the gel; however, the spots were smeared and not well-resolved.