Mao Yu, Zhang Xiangmin
Department of Chemistry, Fudan University, Shanghai, China.
Electrophoresis. 2003 Sep;24(18):3289-95. doi: 10.1002/elps.200305578.
A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.
本文描述了一种用于蛋白质和肽图谱分析的综合二维（2-D）分离系统，该系统将毛细管反相液相色谱（cRPLC）与毛细管等电聚焦（CIEF）相结合。作为第一维的cRPLC为无盐蛋白质提供了高分辨率分离。作为第二维的CIEF与cRPLC具有正交机制，对蛋白质具有出色的分辨率能力，并能有效富集蛋白质。由于cRPLC流出物中的所有样品馏分都可以转移到CIEF维度，两种高效分离方法的结合实现了每个维度的最大分离能力。使用复杂的蛋白质/肽样品（如酵母细胞质和牛血清白蛋白胰蛋白酶消化物）证明了该方法的分离效果。实现了超过10000的峰容量。为该系统开发的激光诱导荧光（LIF）检测器实现了高灵敏度检测，对牛血清白蛋白消化物的肽检测水平可达飞摩尔级。使用异硫氰酸荧光素（FITC）和马来酰亚胺硼二吡咯（BODIPY）对蛋白质进行标记，发现在我们的二维系统中，后者在分离和检测方面表现更佳。
