Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized trypsin...

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M-IPG), on-line digestion by trypsin-based immobilized enzyme microreactor (trypsin-IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M-IPG CIEF column and trypsin-IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20  mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M-IPG CIEF, followed by on-line digestion by trypsin-IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE-based on-line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.