As well as advances in procedure from the method by Smithies and Poulik1 to the method pioneered by O’Farrell and Klose4 and the alternative to carrier ampholytes by G�rg et al2, methods in sample preparation have also seen advances and improvements. For a high performance 2DE it is critical that good solubilisation of the protein samples prior to the IEF step is achieved. Traditional methods of sample preparation are very time consuming and laborious.
The method for the solubilisation of the sample usually requires a solubilisation/denaturation (SD) buffer7. This not only separates the proteins but also denatures the protein to ensure reliable running in the IEF phase “Quantitative proteomic analyses have traditionally used two-dimensional gel electrophoresis (2DE) for separation and characterisation of complex protein mixtures. Among the difficulties associated with this approach is the solubilisation of protein mixtures for isoelectric focusing (IEF)”8. A new approach to the method of solubilisation has been developed by using the Taguchi approach, “Taguchi methods are statistical methods developed by Genichi Taguchi to improve the quality of manufactured goods”9. This method has been used for several decades in industry to maximise the process of there products, and is now being applied in the field of science.
To find the optimum conditions for 2DE Khoudoli et al8 applied the principles of the Taguchi method in the formation of the rehydration buffer and optimised the sample reduction and alkylation procedure. The result of applying this method improved the solubility and resolution of protein mixtures derived from a variety of sources on 2DE, “With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved”.
The application of 2DE has been used extensively in the field of neuro-proteomics investigating conditions such as Alzheimer’s and Parkinson’s disease. Sheta et al10 reported that using this technique it enabled the identification of diseased specific serum biomarkers relating to these diseases. The report also outlines the massive potential the technique has for pathway measurement in blood tests for diagnosis, and therapeutic monitoring. The use of 2DE has been utilised in the investigation of human hepatocellular carcinoma-related (HHC) biomarkers11. In an attempt to improve therapy and reduce the mortality of HHC it is important to identify changes in tumour specific biomarkers associated in the progression of this disease.
2DE was used combined with nano flow liquid chromatography tandem mass spectrometry to investigate differentially expressed proteins in HCC successfully and showed that in HHC sixteen proteins seemed to be up regulated and 14 proteins were down regulated. There was a significant up regulation in the 14-3-3 gamma protein in HHC. Isoforms of this protein have been linked to carcinogenesis. “In conclusion, 2D-DIGE is an efficient strategy that enables us to identify differentially expressed proteins in HCC. Identification of potential biomarkers, such as the pinpointing of 14-3-3gamma in our findings, may provide further useful insights into the pathogenesis of HCC” 11.
Conclusion.
In proteomics the use of 2DE has proved to be an invaluable tool in the investigation into disease diagnosis. Through out its introduction it has continued to be the preferred method for protein separation due to its unparalleled ability to separate simultaneously thousands of proteins. I have discussed the changes in the methods and principles of two-dimensional electrophoresis from starch gels to the development of SDS PAGE and the developed protocol of the Taguchi approach in respect to sample preparation. This technique has shown to very effective in investigation of neuro proteomics and HCC which has enabled us to identify chemical biomarkers associated with disease and enables us to monitor therapy more effectively. The use of 2DE in disease diagnosis is enabling us to have better understanding of disease and its process and with the aid of this technique will help us come closer to cures and treatments for a range of diseases.
References.
1. Olof Vesterberg. 1989. History of Electrophoretic Methods. Journal of Chromatography, 480, 3-19.
2. G�rg, A. 2004. 2-D Electrophoresis, principles and methods. 3rd edition. G E Health care.
3. Unknown. Two-dimensional gel electrophoresis. Wikipedia, the free encyclopedia. http://en.wikipedia.org/wiki/2-D_electrophoresis. Last accessed 10/10/06.
4. Pier Giorgio Righetti. 2005. Electrophoresis: The March of Pennies, the March of Dimes. Journal of Chromatography A, 1079, 24-40.
5. Reed R. Holmes D. Weyers J. Jones A. 2003. Practical Skills in Biomolecular Sciences. 2nd edition. Pearson Education.