Rapid proteolytic digestion and peptide separation using monolithic enzyme microreactor coupled with capillary electrophoresis

Proteolytic digestion and peptide separation are key steps in proteomic study, which has attracted much attention. A novel immobilized trypsin microreactor based on monolithic capillary column was developed for rapid proteolytic digestion in this work. The monolithic support was prepared using itaconic acid as functional monomer within a silanized fused-silica capillary. By taking advantage of itaconic acid with two carboxylic functional groups, trypsin was covalently immobilized onto the monolithic support by condensation reaction. The performance of proteolytic digestion by the microreactor was evaluated with cytochrome C and bovine serum albumin, and the digests were further analyzed by CE and HPLC. Compared to those obtained by conventional in-solution digestion, the digestion time was shortened from 12 h to 40 s, demonstrating the high digestion efficiency of the prepared microreactor. Difference of protein separation results obtained by CE and HPLC highlighted the potential of CE in fast and highly efficient peptide separation in proteomic study. All the results demonstrated that the microreactor combined with CE could be a promising tool in workflow of proteomic analysis.