Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, PR China.
J Sep Sci. 2010 Oct;33(20):3194-200. doi: 10.1002/jssc.201000324.
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.
建立了一种由等电聚焦（CIEF）与整体固定化 pH 梯度（M-IPG）分离蛋白质、基于胰蛋白酶的固定化酶微反应器（trypsin-IMER）在线酶解、以及 CZE 分离肽段组成的集成平台。在该平台中，三通接头不仅用于连接 M-IPG CIEF 柱和 trypsin-IMER，还用于提供调节缓冲液以改善蛋白质分离和酶解的兼容性。另一个接口由带有缺口的特氟隆管制成，通过短毛细管将 IMER 和 CZE 连接起来，短毛细管浸入装满 20mmol/L 谷氨酸的离心管中，用于交换蛋白质酶解缓冲液并保持电接触以进行肽段分离。在最佳条件下，通过该平台，核糖核酸酶 A、肌红蛋白和 BSA 的混合物通过 M-IPG CIEF 分离成 12 个馏分，然后通过 trypsin-IMER 在线酶解和 CZE 分离肽段。用高紫外信号观察到许多对应于不同蛋白质的胰蛋白酶肽段峰，表明该集成系统具有优异的性能。我们希望本文开发的基于 CE 的在线平台能够为蛋白质的集成分析提供另一种强大的选择。
