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अमूर्त

An Effective Method for the Analysis of Human Plasma Proteome using Two-dimensional Gel Electrophoresis

Yasmin Ahmad and Narendra Sharma

Proteome analysis of plasma is increasingly leading to biomarker discovery of human diseases. However the highabundant proteins, excess of salt and lipid in plasma makes the analysis very challenging. Therefore it is necessary to improve the sample preparation procedures before/after the two-dimensional gel electrophoresis analysis of plasma proteins. The objective of this study was to develop a reproducible method by examining the following parameters: (1) depletion of the high-abundant proteins (2) effect of different precipitation methods (3) comparing optimised rehydration buffer using modified Taguchi method with the standard rehydration buffer and (4) comparing the effects of different staining methods. Our results showed that the depletion of two high-abundant proteins improved the visualization of less abundant proteins present in human plasma and precipitation with TCA/acetone resulted in an efficient sample concentration and desalting. We found that using optimized rehydration buffer as compared to standard rehydration buffer increased protein solubility, improved resolution and reproducibility of 2D gels. We also found that visualization of 2D gel profiles by silver staining and fluorescent staining enhanced the detection of low abundant plasma proteins as compared to Coomassie staining. In conclusion, the optimized conditions in our study can be applied to produce a better reference 2-DE gel of plasma samples for the identification of novel disease markers.