| Proteomics, defined as the large-scale analysis of proteins, will contribute greatly to the understanding of gene function in the post-genomic era. Profound impact will be made on disease related research from early diagnosis to the identification of targets for drug screening and development. However, comprehensive proteomic analysis is not yet a straightforward objective; the vast numbers of proteins present in the proteome of a typical organism require that some form of separation or pre-fractionation technique be performed on the mixture prior to introduction into the mass spectrometer.; To this end, a gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field-amplification and sample stacking during the extraction process.; Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and β-galactosidase (118 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 1 ng. The SDS-β-galactosidase complexes extracted from a 1 ng protein loading are concentrated in a 20 nL solution plug inside the capillary with an estimated concentration of 0.044 mg/ml or 10−7 M. The capillary format further allows easy integration with miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification.; The major milestone demonstrated is the efficient coupling of electronic gel protein extraction with the μ-trypsin membrane reactor for on-line proteolytic digestion, and MS analysis of protein mass loadings at the limit of visual detection. In comparison with techniques described elsewhere, the electronic gel protein extraction system has the ability to identify proteins resolved by 2D-PAGE at levels at least 50X lower, encouraging the use of this platform toward the sensitive analysis of low abundant proteins in gel resolved systems. |
