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Discrimination of sulfated isomers of chondroitin sulfate disaccharides by HILIC chromatography and on-line coupled tandem mass spectrometry
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Chondroitin sulfate (CS) are sulfated polysaccharides in particular present in the extracellular matrix of the central nervous system. In this study, we developed a HILIC-MS coupling enabling differentiation of both positional isomers and anomers of mono- di- and trisulfated CS disaccharides. The mobile phase composition in terms of pH and concentration showed high influence on chromatographic separation and was varied to allow the distinction of each CS without signal overlapping in 25 minutes total. This methodology was applied to identify the disaccharides isomers resulting from the depolymerization by the chondroitinase lyase ABC on endocan and the enzymatic kinetic of CS 4-O-endosulfatase. We have determined that the depolymerization of endocan yielded 99% of monosulfated disaccharides CS-A/CS-C in a 71/29 ratio, and trace amounts of disulfated CS-D, CS-E and unsulfated CS-0 disaccharides. In addition, the off-line desulfation kinetic performance of CS 4-O-endosulfatase on the monosulfated CS-A disaccharide allowed determining the half-time of the reaction at 4.5 ± 0.9 min in the experimental setup
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