Ultrasensitive analysis of glycosaminoglycans by capillary electrophoresis coupled with laser induced fluorescence detection

Abstract

Rapid and high-sensitive determination of carbohydrate components of glycoconjugates is critical for the advancement of glycobiology. However, conventional methods pose great challenges because of the low concentration of carbohydrate contents from many biological samples and lack of natural chromophores or fluorophores for robust analysis. Here we present two examples of using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection for ultrasensitive analysis of glycosaminoglycans (GAGs).

Fluorescence resonance energy transfer (FRET) is commonly used in DNA assays, in protease assays and in protein structural biology, but FRET is infrequently applied to carbohydrate analysis due to the lack of active sites on a carbohydrate molecule and proper pathways to assemble a FRET pair into a close proximity to produce efficient FRET. We developed a method based on a quantum dot (QD)-Cy5 hybrid FRET pair for carbohydrate analysis that is capable of detecting very low concentrations of disaccharides. QDs also function as a concentrator of target disaccharides and increase the overall FRET efficiency. Unlinked QDs and Cy5-hydrazide produce a near-zero background signal on CE-LIF with two different band-pass filters. With this simple model, we studied the quantitative properties of this FRET system. This method can be applied to ultrasensitive analysis of disaccharide analysis and potentially applied to that high-throughput analysis of GAGs.

Quantitative disaccharide compositional analysis is one of the most widely used strategies for structural characterization of GAGs, which is directly related to understanding their biological functions. Current methods of HP/HS-derived, CS/DS-derived and HA-derived disaccharides analysis are completed in separate chromatographic or electrophoretic experiments, which calls for time-consuming multiple sample recoveries and separate analysis steps. To solve this problem, we developed a CE-LIF method to resolve seventeen AMAC-labeled GAG-derived disaccharides in a single run with attomolar detection level and applied it to biological samples. This method can also simplify conventional enzymatic degradation procedure.