The Phase I of this project aims to demonstrate the combined usage of innovative technologies which provide the critically needed link between 2-D PAGE and MALDI-MS, namely electronic protein transfer and nanoscale proteolytic digestion in an integrated and high throughput platform. Our research efforts will develop and validate the technical basis for effectively extracting negatively charged SDS-protein complexes from polyacrylamide gel into fused-silica capillary, followed by ultrafast in situ tryptic digestion and deposition onto the MALDI target for protein identification. Our research objectives are to provide the capability to identify low abundance proteins by achieving field-amplification protein stacking/concentration during the protein extraction step and performing in-capillary proteolytic digestion, effectively avoiding analyte loss and dilution. The newly developed trypsin membrane reactor enables comprehensive proteolytic digestion in seconds instead of hours, while offering capillary interfaces for on-line combination with gel protein extraction and direct deposition of protein digest onto the MALDI target. In order to implement these technologies in a commercial instrumentation platform for automated and high throughput analysis, a number of system-level issues will be addressed in the Phase II of this research.
| Cooper, Jonathan W; Gao, Jun; Lee, Cheng S (2004) Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometry. Electrophoresis 25:1379-85 |
