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Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis
Author(s) -
Wang Tingting,
Ma Junfeng,
Zhu Guijie,
Shan Yichu,
Liang Zhen,
Zhang Lihua,
Zhang Yukui
Publication year - 2010
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201000324
Subject(s) - chromatography , chemistry , trypsin , capillary electrophoresis , isoelectric focusing , microreactor , capillary action , immobilized enzyme , buffer (optical fiber) , immobilized ph gradient , biochemistry , enzyme , materials science , telecommunications , computer science , composite material , catalysis
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.