Two-dimensional (2D) gel electrophoresis is a powerful technique enabling simultaneous visualization of relatively large portions of the proteome. However, the well documented issues of variation and lack of sensitivity and quantitative capabilities of existing labeling reagents, has limited the use of this technique as a quantitative tool. Two-dimensional difference gel electrophoresis (2D DIGE) builds on this technique by adding a highly accurate quantitative dimension. 2D DIGE enables multiple protein extracts to be separated on the same 2D gel. This is made possible by labeling of each extract using spectrally resolvable, size and charge-matched fluorescent dyes known as CyDye DIGE fluors. 2D DIGE involves use of a reference sample, known as an internal standard, which comprises equal amounts of all biological samples in the experiment. Including the internal standard on each gel in the experiment with the individual biological samples means that the abundance of each protein spot on a gel can be measured relative (i.e. as a ratio) to its corresponding spot in the internal standard present on the same gel. Ettan DIGE is the system of technologies that has been optimized to fully benefit from the advantages provided by 2D DIGE.

Fluorescence two-dimensional differential gel electrophoresis (2-D DIGE*) is a new development in protein detection for two-dimensional gels. Using mouse liver homogenates (control and paracetamol (N-acetyl-p-aminophenol, APAP)-treated), we have determined the quantitative variation in the 2-D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster 2D-Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter-animal response variability in inbred mice was approximately nine times that contributed by the 2-D DIGE process. A number of the most frequently observed protein changes resulting from APAP-treatment were identified by mass spectrometry. Several of these can be rationalised based on available data on the mechanism of APAP hepatotoxicity but others cannot, indicating that proteomics can provide further insights into the biochemical basis of APAP toxicity.

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Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

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2‐D Difference Gel Electrophoresis (DIGE) is a novel technique that enables multiple protein extracts to be labeled with different fluorescent dyes. The labeled samples are then separated on the same 2‐D gel. The dyes known as CyDye TM DIGE Fluors are spectrally resolvable as well as size‐ and charge‐matched. The ability to add more than one sample to a 2‐D electrophoresis (2‐DE) gel enables the introduction of an internal standard that adds the quantitative advantages of a reference sample commonly seen in DNA microarray studies. Measurement of protein abundance as a “Standardized Ratio” instead of an “Absolute volume” greatly reduces the influence of experimental variation commonly associated with conventional 2‐DE.