Differential in-gel electrophoresis

Zhou G, Li H, DeCamp D et al. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol Cell Proteomics 2002 117-123. 

DGE has been sometimes substituted by differential in gel electrophoresis (DIGE) technique to prevent gel-to-gel irreproducibility. In DIGE, different samples are labeled with ultrahigh-sensitive fluorescent dyes, typically Cy5 and Cy3, and then loaded in the same gel. After separation, gel images... 

Differential in-gel electrophoresis (DIGE) facilitates protein expression by labeling different populations of proteins with fluorescent dyes. Typically, paired samples from the normal and tumor region are labeled with Cy3 and Cy5. After analysis by differential analysis image software, protein spots that exhibit a significant difference in intensity are excised for in-gel tryptic digestion and MS analysis. 

D Differential In-Gel Electrophoresis (DIGE). DIGE this is a modification of 2D gel electrophoresis to avoid any differences that are usually encountered when samples are run on different gels even under identical conditions. In this method, two protein samples with the same amount of protein in each are linked covalently at lysine residues with fluorescent cyanine dyes cy3 and cy5. The two samples are then mixed and run on the same gel, and then the separated proteins are visualized by excitation of cy3 or cy5 specific fluorescence imaging. The differences in the protein bands of normal and cancer cells have been visualized in this way. 

Inner medullary collecting ducts (IMCD) isolated from rats treated with lithium for either 1 or 2 weeks were subjected to differential 2D gel electrophoresis combined with mass spectrometry and bioinformatic analysis to identify signahng pathways affected by lithium indicated that proteins involved in cell death, apoptosis, cell proliferation, and morphology are highly affected by lithium. Lithium treatment increased the intracellular accumulation of (3-catenin in association with increased levels of phosphorylated glycogen synthase kinase type 3p (GSK3(3) [61]. 

D-differential imaging gel electrophoresis (DIGE) is also a gel-based procedure, but one that is more precise than the 2-DE approach. A key element of this procedure is labeling of proteins in the two experimental samples with different colored amine reactive fluorescent dyes.46 47 A common procedure is to label the control sample with Cy-5 cyanine dye and the test sample with Cy-3 cyanine dye. These two dyes display fluorescence at 670 and 570 nm, respectively. The two samples are then mixed in equal concentrations and separated... 

Quantification by Two-Dimensional Differential Imaging Gel Electrophoresis 2D-DIGE is a more precise version of the 2-DE approach. This method uses multicolored cyanine dyes to label proteins in the two experimental samples... 

Since it is possible to differentiate well-preserved from badly preserved collagen through amino acid analysis and gel electrophoresis, it is also possible to determine which bone samples are likely to give erroneous isotopic ratios. At least for 8 C, it should be possible to estimate the in vivo isotopic signature by correcting the changed amino acid concentrations of the collagen extract. This way, a reasonable approach to the reconstruction of pale-odiet should be possible. 

Figure 3.1. Protein expression mapping using 2-D electrophoresis and mass spectrometry. The purpose is to compare protein expression patterns between cell types or in the same cell type under different growth conditions. Proteins are extracted from the different cell types and separated by 2D gel electrophoresis. Image analysis programs are used to compare the spot intensities between gels and identify proteins that are differentially expressed. The protein of interest is excised from the gel and its identity is determined by mass spectrometry. The power of the method increases greatly if the identity of a large number of proteins on the gel is known and present in a database because information can then be obtained without further mass spectrometry.

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

Second, the technology has mediocre reproducibility. Software is available to morph images so that spots can be lined up such software is expensive, difficult to use, and not always accurate in its alignment. To overcome this problem and to simplify quantitative comparisons between samples, Unlu et al. (1997) developed differential gel electrophoresis (DIGE), where two samples are each labeled with different fluorescent tags, pooled, separated on the same gel, and scanned at characteristic wavelengths to resolve the components. This technology has been commercialized by Amersham. 

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

CE provides analysis based on orthogonal separation principles compared to other techniques as well as high resolving power. Like slab gel electrophoresis, CE is a family of techniques that resolve sample components by differences in intrinsic molecular characteristics such as size, mass, charge, differential interaction, and isoelectric point (pi). 

In this group of methods the sample constituents can migrate differentially in zones through the capillary in a medium that can be either a gel (CGE) or an electrolyte (free solution CE). The experimental setup of commercial CE systems for zone electrophoresis is similar to the one presented in Figure 1. 

---