Development of?a sensitive?method to measure heparan sulfate 6-o-endosulfatase - the proposal is aimed to develop a sensitive method to measure the activity of heparan sulfate 6-o-endosulfatase (sulf)

Sulf is an enzyme that removes the 6-o-sulfo groups from heparan sulfate polysaccharides to modulate the functions of heparan sulfate. Evidence has demonstrated that the level of sulf contributes to the progression of cancers; however, the method that measures the activity from biological sources is lacking. In this phase i sbir proposal, we propose to use a LC-MS/MS method to determine the activity from plasma and solid tissues. The crucial innovation is that we will use structurally homogeneous heparan sulfate oligosaccharides as substrates to address the issue for the selectivity of the measurement. In addition, we plan to use 13C-labeled oligosaccharide substrates and coupled with disaccharide analysis using a LC-MS/MS, which is expected to increase the sensitivity and add quantitation capability. Aim 1 is designed to find the optimal oligosaccharides for purified sulf enzyme, demonstrating the feasibility of using oligosaccharides to measure the activity of sulfs, including both sulf-1 and sulf-2. We plan to test different oligosaccharides in the size ranging from hexasaccharide (6-MER) to octasaccharide (8- MER). Aim 2 is improve the quantification accuracy using 13C-labeled HS oligosaccharide substrates and LC-MS/MS. Here, we plan to use 13C-labeled oligosaccharide substrate in the analysis. The use of 13C-labeled substrate will eliminate the interference from endogenous HS. Instead of directly measuring DE-sulfated oligosaccharide, we plan to degrade the 13C-labeled oligosaccharides into disaccharides using heparin lyases followed by LC-MS/MS analysis. We expect that disaccharide analysis will significantly increase the sensitivity compared to the measurement of intact oligosaccharides, offering a sensitive and quantitative method to measure the activity of sulf from tissues and plasma. In the phase II analysis, we plan to expand the analysis to a wide range of cancer tissues. The success will lead to a necessary tool for glycoscience to investigate the roles of heparan sulfate in cancers.