Analytical developments and biochemical applications of capillary electrochromatography/mass spectrometry.

摘要

Capillary electrochromatography (CEC) combines the best features of microcolumn liquid chromatography (μ-LC) and capillary electrophoresis (CE). The separation mechanism is mainly chromatographic, while the mobile phase is driven through the stationary phase in a packed capillary column by electroosmotic flow. The method offers high separation efficiency and resolution for complex mixtures within short analysis times.; We have developed a one-step method for the in-situ preparation of macroporous polyacrylamide/poly(ethylene glycol) based CEC column matrices for capillary electrochromatographic separations. Since the polymeric matrix is covalently attached to the wall of the capillary, the resulting monolithic columns are stable without frits. This type of a column provides an easy modification of various functionalities on the gel, while the stabilities of gel are experienced over wide range of pH values and percentage of organic solvents. During the recent past, we have developed and applied hydrophobic and hydrophilic columns to the electrochromatographic separations of steroids and bile acids in biological matrices. Gradient elution systems have also been designed to improve the component resolution.; Glycosylation is one of the most important post-translational modifications of proteins. The oligosaccharide residues play important functions in a variety of biological processes. An increasing need to understand the functions of glycoproteins has stimulated intensive study toward characterizing the glycans of a glycoprotein and elucidating complex structures of N-linked (asparagine residues) and O-linked oligosaccharides (serine and threonine residues). After we developed the hydrophilic-phase CEC columns featuring amino and cyano functionalities, we become successful in separating a wide range of neutral saccharides: mono- and disaccharides, saccharide derivatives, various types of isomers, and glycoprotein-derived oligosaccharides. Coupling of CEC to different mass spectrometry (MS) systems has provided us with the separation and on-line characterization of complex saccharide mixtures. The sheath liquid was optimized to provide high-sensitivity detection and facilitate simple and informative tandem mass spectra. The on-line collision-induced dissociation experiments offered structural elucidation. The ion-trap and Fourier-transform mass spectrometers have been successfully coupled to CEC for the on-line characterization of neutral glycans.