A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2-DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidl y becoming the premier tool for protein identification. When combined, 2-DE and MS form the current operating paradigm for classical proteomics. One o f the key challenges of proteome research is that of detecting and identify ing all of the elements (proteins) of a proteome. Silver staining and radio labelling, e.g. with S-35-methionine ([S-35]-met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total pro tein loading on a two-dimensional (2-D) gel and challenges protein identifi cation using current MS-based technology Therefore, it is necessary to refe r to and locate a radiolabelled spot's cognate on a preparatively loaded st ained gel, or Western blot, and use that protein spot for identification. U nfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to acc urately locate and select spots of interest by visual comparison. We have e stablished a technique that permits the unambiguous localisation of radiola belled proteins on the same silver stained 2-D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and datab ase searching using matrix-assisted laser desorption/ionization-time of fli ght mass spectrometry and by peptide sequencing using tandem MS by hybrid q uadrupole/orthogonal acceleration time of flight MS (Q-TOF).