Mass spectrometric protein identification from two-dimensional gel separation with stain-free detection and visualization using native fluorescence

Abstract

We describe here an approach for the mass spectrometric identification of proteins in proteome analysis from 1D- and 2D-gel electrophoretic separation, using stain-free detection and visualization based on native fluorescence. Staining procedures such as by Coomassie Brilliant Blue, silver salts and fluorescent dyes are typically employed to visualize gel-separated protein bands with high detection sensitivity, however all of these staining procedures produce significant background in mass spectrometric analysis. Using the native fluorescence of aromatic protein amino acids with UV transmission at 343 nm as a fast gel imaging system, unstained visualized protein spots were localised. Upon excision from gels using precise spot picking tools, gel spots were proteolytically digested and analysed by matrix-assisted laser desorption-ionisation mass spectrometry (MALDI-MS). After initial development and testing using 1D-gel separated standard proteins, the stain-free detection approach was successfully applied to MALDI-MS protein identifications in (i), bacterial proteomics of Desulfotignum phosphitoxidans, and (ii), in porcine skeleton muscle proteomics. Major advantages of the stain-free gel detection approach are (i), rapid analysis of proteins from 1D- and 2D-gel separations without destaining required before proteolytic digestion; (ii), low detection limits of proteins in gels; and (iii), low background in the mass spectrometric analysis of proteins.