Novel Proteomics Tools Based on Two-Dimensional Difference Gel Electrophoresis (2D-DIGE)

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Two-dimensional difference gel electrophoresis (2D-DIGE) is a major proteomics tool in the project. 2D-DIGE is an advanced version of classical two-dimensional gel electrophoresis (2D-PAGE). The introduction for 2D-DIGE and its possible applications in cancer proteomics will be described.

Two-dimensional gel electrophoresis (2D-PAGE)
2D-PAGE separates proteins according to their isoelectric point first and molecular weight. As isoelectric point and molecular weight are independent physical characters of proteins, the separation performance of 2D-PAGE is substantially high, next to mass spectrometry. Elcctrophoretic mobilities of proteins reflect the posttranslational modifications (PTMs) because the PTMs such as phosphorylation and glycosylation affect the isoelectric point and molecular weight of the proteins. Therefore, 2D-PAGE can detect the disease-associated aberrations of PTMs. The size (area and intensity) of protein spots changes in parallel with expression level of proteins, and thus 2D-PAGE can achieve quantitative comparison between multiple samples.


Figure 1 Principal of 2D-PAGE. The proteins are separated according to their isoelectric point first and molecular weight.

The technical innovation in 2D-PAGE was the development of Immobiline DryStrip gel. Immobiline DryStrip gels contain a pre-formed pH gradient immobilized in a homogeneous polyacrylamide gel. Immobiline DryStrip gel with various isoelectric ranges and gel length are commercially available. In Immobiline DryStrip gel, the pH gradient is stable and relative large amount of proteins can be loadable. In addition, it is constantly commercially available. With these advantages, Immobiline DryStrip gel is the most popular reagents for isoelectric focusing in 2D-PAGE.

One of the unique advantages of 2D-PAGE is that the proteome data can be obtained in the way that data can be stored in database. Various proteome database based on 2D-PAGE are published for different species, organ, tissues, cells and diseases.

Two-dimensional difference gel electrophoresis (2D-DIGE)
In 2D-DIGE, the protein samples are labeled with fluorescent dyes and then separated by 2D-PAGE. Different protein samples are labeled with different fluorescent dyes, mixed together and separated by the identical gels. The gels are scanned by laser scanners and the image of 2D-PAGE for multiple samples are obtained from single gels. Gel-to-gel variations are the most severe problem in the gel-based proteomics. However, 2D-DIGE solves this problem by resolving the multiple samples in single gels. Figure 2 demonstrates the basic protocol of 2D-DIGE. The different samples as many as the number of fluorescent dyes can be studied in single gels.


Figure 2 Basic protocol of 2D-DIGE. Multiple samples are labeled with different fluorescent dyes, mixed together and separated by single gels.

The advanced protocol enables the comparison between the samples more than the number of fluorescent dyes. Internal control sample, which is a mixture of small portions of individual samples, is created and labeled with Cy3 fluorescent dye. The individual samples are labeled with Cy5 fluorescent dye. These differently labeled proteins are mixed together and separated by the individual gels (Figure 3). The Cy3 laser scanning generates the image of the internal control sample and the Cy5 laser scanning generates the image of the individual samples. All gels can generate the Cy3 images. The internal control sample includes all proteins in the individual samples, as it is a mixture of all samples, and the Cy3 image of the internal control sample includes all protein spots of the individual samples. Normalization of Cy5 intensity with Cy3 intensity cancels out the gel-to-gel variations. This protocol enables the comparative study of several ten and hundreds samples using two types of fluorescent dye, Cy3 and Cy5. The classical 2D-PAGE cannot have internal control for all protein spots simultaneously. The simple idea to label the proteins with fluorescent dye before gel electrophoresis enable these experiments.


Figure 3 Protocol to compare the multiple samples by 2D-DIGE.

Fluorescent dyes used in 2D-DIGE have much more sensitivity than silver staining. For instance, using CyDye DIGE Fluor saturation dye, proteomic study can be achieved using only one ƒÊg amount of proteins. With CyDye DIGE Fluor saturation dye, 2D-PAGE can be achieved using a minute amount of proteins such as those obtained by laser microdissection. The application in which the samples obtained by laser microdissection and labeled with high sensitive fluorescent dyes was developed by the project, and many researchers over the world use it (reference)


Figure 4 Application of Cydye DIGE Fluor saturation dye for laser microdissected tissue samples. The proteins extracted from the cells recovered by laser microdissection are labeled with CyDye DIGE Fluor saturation dye and separated by 2D-PAGE (Kondo et al, Proteomics 2003)

A large format 2D gel device developed in the project separates the labeled proteins. The resolution of protein spots depends on the size of gel area, and the larger gel can generates more protein spots and more accurate proteome data. Image analysis software cannot reproducibly recognize the aggregated protein spots, and in this sense, the large gel has an advantage on the smaller gel too.


Figure 5 Large format gel electrophoresis devices. Gradient makers and electrophoresis devices with a cooling syetem (Bio Craft). The size of glass plates are adjusted to the maximum area of laser scanners (Kondo et al, Nature Protocols 2006)

In 2D-DIGE, the gel staining is replaced by laser scanning. The colorimetric staining methods such as silver staining require much pure water, labor intense and a large working area. CBB staining is a simple way of staining but its sensitivity is not high enough. In contrast, 2D-DIGE images are obtained within one hour by simple laser scanning. The project has been running approximately 2,000 gels annually using multiple laser scanners.


Figure 6 Use of multiple laser scanners enables high throughput proteomics (Kondo et al, Nature Protocols 2006). The project uses six laser scanners.

The images obtained by 2D-DIGE are analyzed by the image software such as DeCyder software and Progenesis SameSpots. The normalization of Cy5 intensity with Cy3 intensity is automatically achieved for all protein spots by the software. The progress of image analysis software is amazing and the price was dramatically reduced. For the data-mining approach to integrate the proteome data and clinico-pathological information, the data about protein spots are exported out from the software for further study by data-mining software such as Expressionist. The data-mining software, ProteoMiningSuite, for 2D-DIGE is under development by a collaboration with Infocom corporation.

Mass spectrometry identifies the proteins corresponding to the protein spots. The proteins in the gels are treated with trypsin and extracted as a mixture of peptides (In-gel-digestion method). The project optimized this method so that proteins for almost all protein spots can be identified by mass spectrometry (Kondo et al, Nature Protocols 2006). Maintenance of mass spectrometry machine is also crucial for protein identification. Optimization of the in-gel-digestion and well-maintained mass spectrometry is a key for sensitive protein identification.


Figure 7 Overall workflow of protein identification (Kondo et al, Nature Protocols 2006)

4. 2D-DIGE with the other experimental techniques
Combination of 2D-DIGE with the other experimental techniques is an effective approach for more comprehensive proteomic studies. For instance, chromatographic separation of plasma samples prior to 2D-DIGE increases the number of protein spots in plasma proteomics. This method was applied to the studies on lung cancer and pancreatic cancer. In general, protein fractionation prior to 2D-DIGE can improve the separation performance and effective for plasma proteomics.