Comparative fluorescence two‐dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on‐gel generation of a reference protein spot grid

Abstract

The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.

Horizontal comparative fluorescence two‐dimensional gel electrophoresis for improved spot coordinate detection

“…In case of samples, which are available only once such as urine or blood from a patient or samples collected in field work or for occupational workplace testing, the methodology cannot be applied. Therefore, we introduced comparative fluorescence 2D gel electrophoresis (CoFGE, ), a vertical 2DE method that improved the mean deviation of x ‐ and y ‐coordinates of protein spots in independent experiments from more than 7% to about 1%.…”

“…Early CoFGE ignored common wisdom in two points . First, variability due to the p I of the proteins was not taken into account; the method relied on high‐quality commercial p I strips that were placed at precisely the same position on the gel.…”

“…15) to manually remove the gel plugs. These wells of 1 mm id held 0.5 μL of reference protein mix (homemade composition consisting of nine commercially available proteins with molecular masses from 8.6 to 97 kDa ).…”

“…The equipment for 2DE was from GE Healthcare (Freiburg, Germany; DryStrip Reswelling Tray, Ettan IPGphor II Manifold, Typhoon 9400, ImageQuant software) and Serva (Heidelberg, Germany; HPE FlatTop Tower, PaperPools, FS wicks, cooling contact fluid, SERVA IPG Blue Strip 24 cm pH 4–7, electrode and equilibration buffer). The 2DE experiments were performed in the first dimension using Ettan IPGphor II (maximum current set to 50 μA per strip) and High Performance Electrophoresis FlatTop Tower for commercially available gels (255 mm × 200 mm × 0.65 mm) in the second dimension (14.5°C). Protein in sample buffer was cup‐loaded onto the strip.…”

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

“…Quantitative proteomics allows the comparison of distinct proteomes to identify proteins that display changes in abundance or post‐translational state. 2D DIGE technology is a 2D gel technology that relies on pre‐electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, and mixing of these labeled samples prior to their electrophoretic separation on the same gel, and this technique is widely used . Recently, this technique has been applied for identifying biomarkers, designing novel drug targets and monitoring therapeutic processes.…”