On-Line Hyphenation of Capillary Isoelectric Focusing and Capillary Gel Electrophoresis by a Dialysis Interface

Abstract

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the seconddimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has been a standard technique for high-throughput separation and characterization of biological macromolecules, especially proteins. A main advantage of 2D PAGE is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. 1 More than one sample can be dealt with in a single 2D PAGE run, but a total 2D PAGE process usually lasts for at least 1 day and all the manual operations are time-consuming and laborsome. Capillary electrophoresis (CE) is an easier and more rapid alternative to slab electrophoresis. CE was thoroughly reviewed 2-7 and now is a powerful tool for the separation of proteins and peptides. [8][9][10][11][12][13] Several kinds of CE mode are built based on different principles. Each single CE mode has its distinct capabilities and limitations. To take full advantage of the capabilities and to avoid the limitations of CE modes it is helpful to combine them one to another [14][15][16][17][18][19][20][21][22] or with separation methods based on other than electromigration principles. Much attention bas been paid to the hyphenation of CE with liquid chromatography (LC). [23][24][25][26][27][28][29][30][31] There has been a great deal of effort to interface CE with mass spectrometry (MS); nonetheless, MS was considered as a molecular weight detector rather than a separation tool. [32][33][34][35][36][37][38] * Corresponding author: (e-mail) ykzhang@dicp.ac.cn; (phone) +86-411-3693427; (fax) +86-411-3693427.(1) Lilley, K. S.; Razzaq, A.; Dupree, P. Curr. Opin. Chem. Biol. 2002, 6, 46-50. (2)

Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation

“…Micellar electrokinetic chromatography has been coupled to CZE 10,11 or capillary gel electrophoresis (CGE). 12 In both cases, high-resolution separations have been obtained in a reduced period of time. However, in the context of developing two-dimensional separations, CIEF, due to its high loading capacity, resolution, and concentrating power, appears as the most suitable first dimension.…”

“…However, in the context of developing two-dimensional separations, CIEF, due to its high loading capacity, resolution, and concentrating power, appears as the most suitable first dimension. So far, CIEF has been coupled to CGE 12 and to CZE. 10 A very accomplished work is certainly the one published by Mohan et al 13,14 In these studies, a microdialysis junction has been employed to hyphen CIEF and CZE.…”

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

“…Zhang and co-workers combined CIEF with capillary gel eletrophoresis (CGE) [38], by mounting a hollow fiber on a methacrylate resin plate (Fig. 1).…”

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

“…Capillaries (50-lm ID, 375-lm OD) from Ruiyang Chromatographic Device Co. Ltd (Yongnian, Hebei, China) were coated with polyacrylamide as described previously [16]. In brief, after being cleaned and dried a capillary was filled with a solution of c-MAPS (50% v/v in methanol) and kept at room temperature with both the ends sealed by silicone septa.…”