Improved Workup for Glycosaminoglycan Disaccharide Analysis using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

摘要

This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan disaccharide analysis from complex biological samples. In the process of applying capillary electrophoresis with laser-induced fluorescence to glycosaminoglycan (GAG) disaccharide analysis in biological samples, we have made improvements to existing methods. These include (1) optimization of reductive amination conditions, (2) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization and, (3) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1 pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate disaccharides or eleven of twelve standard heparin/HS disaccharides, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV with 0.3 psi pressure. Relative standard deviation in migration times of CS ranged from 0.1% to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem mass spectrometry. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/HS and CS/DS disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue.