Quantitative analysis of heparan sulfate using 13C-labeled standards

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This SBIR phase I proposal is aimed at developing to develop a LC-MS/MS based method to analyze heparan sulfate in the sub-nanogram range. Heparan sulfate is a highly sulfated polysaccharide displaying a wide range of biological functions. There is a strong demand for a quantitative and sensitive method to analyze heparan sulfate. In this proposal, we propose to achieve the quantitation of heparan sulfate using the disaccharide analysis. To this end, heparan sulfate is depolymerized into eight disaccharides by heparin lyases. The composition analysis of the resultant disaccharides is accomplished by a LC-MS/MS method. The crucial innovation in our proposed method is to add “absolute quantitation” capability using eight 13C-labeled disaccharide calibrants and one 13C-labeled polysaccharide calibrant. Aim 1 is designed to demonstrate the ability to quantitative compositional analysis of heparan sulfate from mouse tissues and plasma. In phase I studies, we will focus on analyzing the HS from plasma, lung, liver, and kidney from healthy mice and burn injury mice. Aim 2 is aimed to conduct quantitative analysis of heparan sulfate from a single histological slide. The sensitivity of the LC-MS/MS method will be improved. In the phase I studies, we will analyze the heparan sulfate from histological slides from lung, liver, and kidney from healthy and burn injury mice. In the phase II studies, we plan to expand the analysis of heparan sulfate to all mouse tissues from healthy mice and four disease mice. We also plan to conduct the analysis from clinical samples from burn patients and sepsis patients in phase II studies. The success of this project will provide a method to analyze heparan sulfate with sensitivity and accuracy for biologists and will be a major break-through in glycobiology research.