A 2D-difference gel electrophoresis strategy for redox proteomics
Post-genomic biomedical science requires quantitative proteomics. In most cases this involves differential protein expression analysis using matched pairs of simultaneously detectable labelling reagents for specific protein amino acids. In this thesis the development and optimisation of a novel cysteine labelling strategy, that is based on the use of iodoacetyl derivatives of Cy3 and Cy5 (ICy3/5) and 2D-difference gel electrophoresis (2D-DIGE) is described. The differentially labelled samples are separated on a single 2D gel and detected by multi-wavelength fluorescence scanning. The method is used to analyse standard proteins and then cell lysates to define the stoichiometry, sensitivity and specificity of this labelling technique. A comparative study of this new proteomic ICy dye protocol with the current NHS-Cy dye labelling system and methods that employ commonly used protein staining methods is described. The method is then used for cysteine labelling of proteins in non-reduced, denatured biological samples allowing accurate monitoring and sensitive detection of redox-dependent thiol modifications and expression level changes. The method is shown to be compatible with the use of MALDI mass spectrometry to identify proteins by analysis of trypsinised ICy labelled peptide digests. Using parallel sample analysis within single gels, the ICy-dye reagents were used to detect redox-, ErbB-2- and growth factor-dependent changes in a human mammary luminal epithelial cell system which was exposed to hydrogen peroxide or to growth factor stimulation. The conventional lysine labelling 2D-DIGE technique was also used in parallel to assess the new ICy labelling strategy for determination of the effects of oxidative stress on protein isoform levels. This study has revealed the identity of proteins involved in the response to oxidative stress and growth factor stimulation in the context of ErbB-2 growth factor receptor over-expression. In addition, this labelling strategy was also used to detect changes in thiol reactivity that follow the UV irradiation of plasma proteins as part of a study designed to evaluate the effect of UV disinfection on plasma product safety for clinical use.