Abstract
The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.
Highlights
The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification
Proteomics aims to characterize a large number of proteins extracted from a cell, tissue, or organism so that a global perspective of changes in protein expression can be obtained in a rapid fashion [1, 2]
The number of identified MS/MS spectra obtained with the microwave-assisted protein enzymatic digestion (MAPED) protocol was more than that with the standard protocol. These results indicate that the MAPED in-gel protocol could significantly improve peptide recovery efficiency from gel for protein mixtures compared with standard in-gel digestion protocols
Summary
The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. Equivalent (insolution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research. The abbreviations used are: 2D, two-dimensional; 1D, one-dimenresolve and identify peptide components in the mixture This approach, referred to as shotgun proteomics, affords the advantages of automation and sensitivity but at the loss of information regarding the intact protein. Each slice or spot in the gels is excised, digested, and identified by MALDI-MS or ESI-ion trap-MS Another top-down approach uses chromatofocusing and reverse phase chromatography in an HPLC format to separate proteins (9 –12). Other promising approaches include microwave-assisted protein enzymatic digestion (MAPED) or acid hydrolysis (18 –22)
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