Development of Structural Analysis Techniques for Keratan Sulfate Using Chemical Derivatization and LC-MS/MS

摘要

Keratan sulfate(KS), a class of glycosaminoglycans (GAGs), is a linear, sulfated polysaccharide consisting of repeating disaccharide units with variable 6-O-sulfation on each monosaccharide. Unlike other GAGs such as heparan sulfate (HS), KS contains galactose instead of a uronic acid, causing the distribution of charge to be dictated solely by the position of sulfation(s). KS is found in numerous tissues and affects cell hydration, embryonic development, physiological variations, and wound healing. We have demonstrated sequencing of patterns of modification for GAGs such as heparin/heparan sulfate (HS) and chondroitin sulfate (CS) using chemical derivatization and LC-MSn. However, other GAGs have demonstrated notable sample losses due to beta-elimination at the uronic acid during derivatization, especially for longer oligosaccharices. The unique KS disaccharide repeat structure eliminates issues with derivatization-induced beta-elimination, which should allow sequencing of large oligosaccharides, and perhaps full KS chains. Here, we describe development of these techniques for KS. Here, we detail our initial progress towards isolation and characterization of KS from bovine tissue. We also demonstrate our initial experiments examining the effects of the derivatization process on the disaccharide repeat found in KS. We demonstrate our ability to carry the core disaccharide backbone through the entire derivatization process with very high yield. We also demonstrate successful isolation of bovine corneal KS. Future work in KS depolymerization, derivatization, and LC-MS/MS sequencing is discussed.