Another application of 2-DE is in cell map proteomics. 2-DE is used to map proteins from microorganisms (28, 146), cellular organelles (83), and protein complexes (134). It can also be used to resolve and characterize proteins in subproteomes that have been created by some form of purification of a proteome (26, 35, 38, 83). Because a single 2-DE gel can resolve thousands of proteins (30, 44, 146), it remains a powerful tool for the cataloging of proteins. Many 2-DE databases have been constructed and are available on the World Wide Web (15).

A number of improvements have been made in 2-DE over the years (13, 29). One of the biggest improvements was the introduction of immobilized pH gradients, which greatly improved the reproducibility of 2-DE (20, 59). The use of fluorescent dyes has improved the sensitivity of protein detection (126), and specialized pH gradients are able to resolve more proteins (59). The speed of running 2-DE has been improved, and 2-D gels can now be run in the minigel format (139). In addition, there have been efforts to automate 2-DE. Hochstrasser's group has automated the process of 2-DE from gel running to image analysis and spot picking (156). The use of computers has aided the analysis of complex 2-D gel images (16). This is a critical aspect of 2-DE because a high degree of accuracy is required in spot detection and annotation if artifacts are to be avoided. Recently, a molecular scanner was developed to record 2-DE images (19). Software programs such as Melanie compare computer images of 2-D gels and facilitate both the identification and quantitation of protein spots between samples (171). A recent exciting advance in 2-DE was developed by Minden and coworkers (158). This technology is called difference gel electrophoresis (DIGE) and utilizes fluorescent tagging of two protein samples with two different dyes. The tagged proteins are run on the same 2-D gel, and postrun fluorescence imaging of the gel is used to create two images, which are superimposed to identify pattern differences. The dyes are amine reactive and are designed to ensure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. This technique circumvents the need to compare several 2-D gels. In their original paper, DIGE was used to detect differences between exogenous proteins in two D. melanogaster embryo extracts at nanogram levels (158). Moreover, an inducible protein from Escherichia coli was detected after 15 min of induction. This technology is now commercially available from Amersham/Pharmacia.

However, a number of problems with 2-DE still remain. Despite efforts to automate protein analysis by 2-DE, it is still a labor-intensive and time-consuming process. A typical 2-DE experiment can take two days, and only a single sample can be analyzed per gel. In addition, 2-DE is limited by both the number and type of proteins that can be resolved. For example, the protein mixture obtained from a eukaryotic cell lysate is too complex to be completely resolved on a single 2-D gel (29). Many large or hydrophobic proteins will not enter the gel during the first dimension, and proteins of extreme acidity or basicity (proteins with pIs below pH 3 and above pH 10) are not well represented (59). Some of these problems can be overcome with different solubilization conditions and pH gradients (59). Another limitation of 2-DE is the inability to detect low-copy proteins when a total-cell lysate is analyzed (67, 96, 146). In a crude cell extract, the most abundant proteins can dominate the gel, making the detection of low-copy proteins difficult. It was determined in the analysis of yeast proteins by 2-DE that no proteins defined as low-copy proteins were visible by 2-DE (67). Yet it is estimated that over half of the 6,000 genes in yeast may encode low-copy proteins (58). In mammalian cells, the dynamic range of protein expression is estimated to be between 7 and 9 orders of magnitude (36). This problem cannot be overcome by simply loading more protein on the gel, because the resolution will decrease and the comigration of proteins will increase (36). Because of these limitations, the largest application of 2-DE in the future will probably involve the analysis of protein complexes or subproteomes as opposed to whole proteomes.

Alternatives to electrophoresis. The limitations of 2-DE have inspired a number of approaches to bypass protein gel electrophoresis. One approach is to convert an entire protein mixture to peptides (usually by digestion with trypsin) and then purify the peptides before subjecting them to analysis by MS. Various methods for peptide purification have been devised, including liquid chromatography (95, 106, 174), capillary electrophoresis (55, 155), and a combination of techniques such as multidimensional protein identification (95) or cation-exchange chromatography and reverse-phase (RP) chromatography (120). The advantage of these methods is that because a 2-D gel is avoided, a greater number of proteins in the mixture can be represented. The disadvantage is that it can require an immense amount of time and computing power to deconvolute the data obtained. In addition, considerable time and effort may be expended in the analysis of uninteresting proteins. One of the most exciting techniques to emerge as an alternative to protein electrophoresis is that of isotope-coded affinity tags (ICAT). This method allows the quantitative protein profiling between different samples without the use of electrophoresis (see "Proteomics applications" below).