Development of analytical methods for glycosaminoglycans from biological sources

This SBIR phase II proposal is aimed to develop a LC-MS/MS based method to analyze heparan sulfate and chondroitin sulfate with sub-nanogram quantities. Heparan sulfate and chondroitin sulfate are highly sulfated polysaccharides displaying a wide range of biological functions. There is a strong demand for a quantitative and sensitive method to analyze heparan sulfate and chondroitin sulfate. In this proposal, we propose to achieve the quantitation of heparan sulfate and chondroitin sulfate using 13C-labeled calibrants. To this end, heparan sulfate and chondroitin sulfate are depolymerized into disaccharides by heparin lyases and chondroitin ABCase. The composition analysis of the resultant disaccharides is accomplished by a LC-MS/MS method. In addition, we plan to use 13C-labeled heparan sulfate octasaccharide calibrants to determine the levels of 3-O-sulfated heparan sulfate. The crucial innovation in our proposed method is to add “absolute quantitation” capability using 13C-labeled disaccharide calibrants and 13C-labeled octasaccharide calibrants. Aim 1 is designed to demonstrate the ability to quantitative the level of 3-O-sulfated heparan sulfate. We will focus on analyzing the analysis of 3-O-suflated heparan sulfate from plasma samples and from human brain tissues with neurodegenerative diseases. Aim 2 is aimed to develop a method for quantitative disaccharide analysis of chondroitin sulfate. We will synthesize six 13C- labeled chondroitin sulfate disaccharide calibrants to carry out the studies. We will also conduct the analysis for chondroitin sulfate from human brain tissues from neurodegenerative diseases. The completed heparan sulfate and chondroitin sulfate analysis from plasma and solid tissues will demonstrate the robustness of the method. The success of this project will provide a method to analyze heparan sulfate and chondroitin sulfate with sensitivity and accuracy for biologists.