2D-DIGE Quantitative Proteomics

Proteomics is a discipline that studies the cellular, tissue, or organismal composition and its regulatory rules, with the proteome as the research object. Since the establishment of two-dimensional gel electrophoresis (2-DE) by O'Farrell and Klose et al. in 1975, 2-DE has become the core method of proteomics research due to its high sensitivity and resolution, ease of computer image analysis, and good compatibility with mass spectrometry identification methods.

 

In recent years, the research focus of proteomics has shifted from protein identification to quantification. Quantitative proteomics research can be defined as the study of relative abundance differences between multiple samples. Although some optional or complementary proteomics technologies are being developed, such as isotope affinity tags and multidimensional protein identification technology, two-dimensional electrophoresis remains the only key technology that can simultaneously separate a large number of complex protein mixtures.

 

Traditional two-dimensional electrophoresis 2-DE is difficult to ensure complete consistency in sample preparation, electrophoresis conditions, and gel staining, which easily leads to gel-to-gel variations. This variation leads to poor repeatability of the experiment, low sensitivity, and even possible obscuration of real biological differences or misleading false-positive results. To overcome the shortcomings of the 2-DE technology, Unlü et al. proposed two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) in 1997.

 

The principle of the 2D-DIGE technique is to label protein samples with different fluorescent dyes first, then denature the proteins, and then use a fixed pH gradient gel to separate different pH protein bands based on the charge differences of the proteins. Then, the gel strip is placed on a polyacrylamide gel containing SDS, and the proteins are separated according to the molecular weight of the proteins, and the SDS-PAGE gel images of different proteins are analyzed by multi-channel laser scanning. The main steps are: label the protein samples to be compared with two different fluorescent dyes (Cy2, Cy3, or Cy5); evenly mix the fluorescently labeled samples in equal amounts; use the same internal standard to separate the mixed samples by two-dimensional gel electrophoresis; under a fluorescent microscope, use different excitation wavelengths to detect the electrophoresis results.

 

The Notable Advantages of 2D-DIGE Compared to Traditional 2-DE

1. The adoption of fluorescent labeling technology allows for simultaneous comparison of multiple samples, improving experimental efficiency and accuracy.

2. The introduction of fluorescence labeling allows for more precise separation and detection of proteins, enabling the detection of more subtle protein differences.

3. The introduction of an internal standard better eliminates experimental random errors, avoids differences between different gels, and greatly improves the accuracy and credibility of the results.

4. There is no need for post-electrophoresis fixation or decolorization, reducing the loss of proteins, especially low molecular weight proteins.

5. The digital processing of fluorescent signals makes data acquisition and analysis faster and more accurate.

 

MtoZ Biolabs provides SDS-PAGE and 2D-DIGE electrophoresis services, combined with Thermo Fisher's Orbitrap Fusion Lumos mass spectrometry platform and nanoLC-MS/MS nanoscale chromatography, to provide researchers with one-stop proteomics qualitative and quantitative services.