Capillary electrophoresis for total glycosaminoglycan analysis

Abstract

A capillary zone electrophoresis-laser induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline-separated in less then 25 min. This CZE-LIF method gave good reproducibility both migration time (≤ 1.03% for intra-day and ≤ 4.4% for inter-day) and the peak area values (≤ 5.6% for intra- and ≤ 8.69% for inter-day). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results shows that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run.

Compositional analysis and structural elucidation of glycosaminoglycans in chicken eggs

“…After digestion of bovine corneal KS with keratanase II, two saturated disaccharide standards, 2S KS (Gal6S(1→4)GlcNAc6S) and NS KS (Gal(1→4)GlcNAc6S), could be obtained [28]. The HPLC-MS results showed that in all egg components examined, the disaccharide 2S KS was undetectable (Table 2, Fig.…”

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

“…A capillary zone electrophoresis laser-induced fluorescence detection method [34] was recently developed for the simultaneous analysis of disaccharides derived from all four classes. In this study, we developed a simple method for the simultaneous analysis of 23 internal disaccharides from four GAG classes and 5 nonreducing terminal saccharides from CS using LC-ESI-MS/MS with the SRM mode.…”

Heparan sulfate and heparin interactions with proteins

“…Unfortunately, the experiments that could determine unequivocally the origins of specificity cannot be conducted at present for several reasons, which include the need to test vast numbers of structural variants in their pure forms [52]. It is the ability to separate these species in sufficient quantities when using natural HS as a source, despite some progress [53][54][55][56], rather than the problem of sequencing them per se, which hinders progress. Heparin is often employed experimentally as a proxy for HS on account of its structural similarity ( §1) and is considerably more highly charged than HS.…”