Development of a sensitive method to measure heparan sulfate 6-O-endosulfatase

本研究旨在建立一种灵敏的测定乙酰肝素活性的方法 硫酸6-O-内硫酸酯酶（SULF）。SULF是一种酶，可以去除6-O-磺基 硫酸乙酰肝素多糖中的基团来调节乙酰肝素的功能 硫酸盐有证据表明，SULF水平有助于 癌症的进展;然而，测量生物活性的方法 缺少来源。在本阶段I SBIR提案中，我们建议使用LC-MS/MS 测定血浆和实体组织活性的方法。关键的创新是 我们将使用结构均匀的硫酸乙酰肝素低聚糖作为 衬底，以解决测量的选择性的问题。另外我们 计划使用13 C标记的寡糖底物并与二糖偶联 使用LC-MS/MS进行分析，预计将提高灵敏度并增加 定量能力。目的1是寻找纯化的最佳寡糖 SULF酶，证明了使用低聚糖测量 SULFS的活性，包括SULF-1和SULF-2。我们计划测试不同的 寡糖的大小范围从六糖（6-mer）到八糖（8-mer）。 mer）。目的二是提高13 C标记HS寡糖的定量准确度 在这里，我们计划使用13 C标记的寡糖底物和LC-MS/MS。 在分析中。使用13 C标记的底物将消除来自 内源性HS。我们计划不直接测量脱硫酸寡糖， 使用肝素裂解酶将13 C标记的寡糖降解为二糖 随后进行LC-MS/MS分析。我们希望二糖分析将显着 与完整寡糖的测量相比增加了灵敏度， 提供了一种灵敏和定量的方法来测量SULF的活性， 组织和血浆。在第二阶段的分析中，我们计划将分析范围扩大到 癌组织的范围。这一成功将为糖科学提供必要的工具， 研究硫酸乙酰肝素在癌症中的作用。

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.