Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling.

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.