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

开发一种灵敏的方法来测量硫酸乙酰肝素 6-O-内切硫酸酯酶

• 批准号: 10599763
• 负责人: Guowei Su
• 金额: $ 35万
• 依托单位: GLYCAN THERAPEUTICS CORPORATION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10599763
• 关键词: Address; Animals; Biological; Biological Assay; Cancer Patient; Cancer Prognosis; Cell surface; Clinical; Collaborations; Coupled; Custom; Development; Disaccharides; Enzymes; Glycoproteins; Head and Neck Squamous Cell Carcinoma; Heparin Lyase; Heparitin Sulfate; Human; Human Genome; Individual; Label; Licensing; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Microarray Analysis; North Carolina; Oligosaccharides; Oncogenic; Patients; Phase; Physiological; Plasma; Polysaccharides; Process; Production; Protein Isoforms; Recombinants; Research; Role; Sampling; Services; Site; Small Business Innovation Research Grant; Solid; Source; Structure; Sulfatases; Sulfate; System; Technology; Testing; Therapeutic; Tissues; Universities; anticancer research; cancer biomarkers; cancer diagnosis; cost; design; detector; improved; innovation; method development; phase 1 study; success; tool; tumor microenvironment; tumor progression

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.

该提案旨在开发一种灵敏的方法来测量肝素的活性。 硫酸盐6-O-内硫酸酯酶(Sulf)。硫磺是一种去除6-O-磺酸的酶 硫酸肝素多糖基对肝素功能的调节作用 硫酸盐。有证据表明，硫磺的水平有助于 癌症的进展；然而，从生物学角度衡量活性的方法 消息来源匮乏。在此第一阶段SBIR建议中，我们建议使用LC-MS/MS 方法从血浆和固体组织中测定活性。关键的创新是 我们将使用结构均一的硫酸乙酰肝素低聚糖作为 以解决测量的选择性问题。此外，我们 计划使用13C标记的寡糖底物并与双糖偶联 使用LC-MS/MS进行分析，有望提高灵敏度并增加 量化能力。目标1旨在寻找纯化的最佳低聚糖 Sulf酶，论证了用低聚糖来测定蛋白质的可行性。 SULFS的活性，包括Sulf-1和Sulf-2。我们计划测试不同的 低聚糖的大小从六糖(6-聚体)到八糖(8-聚体) Mer)。目的二是利用13C标记HS寡糖提高定量准确度 底物和LC-MS/MS在这里，我们计划使用13C标记的寡糖底物 在分析中。使用13C标记的底物将消除来自 内源性HS。我们不是直接测量脱硫寡糖，而是计划 肝素裂解酶降解~(13)C标记的寡糖为二糖 然后进行LC-MS/MS分析。我们预计双糖分析将显著 与完整低聚糖的测量相比提高了灵敏度， 提供了一种灵敏、定量的方法来测量硫磺的活性 组织和血浆。在第二阶段的分析中，我们计划将分析扩展到广泛的 癌症组织的范围。这一成功将为血糖科学带来必要的工具 研究硫酸乙酰肝素在癌症中的作用。