Quantitative analysis of heparan sulfate using 13C-labeled standards

使用 13C 标记标准品定量分析硫酸乙酰肝素

• 批准号: 10250595
• 负责人: Vijayakanth Pagadala
• 金额: $ 30万
• 依托单位: GLYCAN THERAPEUTICS CORPORATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2022-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250595
• 关键词: Animals; Biological; Biological Process; Blood coagulation; Burn injury; Cell Differentiation process; Clinical; Collaborations; Communities; Coupled; Disaccharides; Disease; Embryonic Development; Ensure; Glycobiology; Goals; Heparin Lyase; Heparitin Sulfate; Individual; Inflammatory Response; Institutes; Kidney; Label; Legal patent; Licensing; Liquid Chromatography; Liver; Lung; Measurable; Measurement; Mediation; Methods; Monitor; Mus; North Carolina; Oligosaccharides; Patients; Phase; Plasma; Play; Polysaccharides; Principal Investigator; Publishing; Reagent; Recovery; Research; Role; Sampling; Services; Slide; Small Business Innovation Research Grant; Source; Structure; Sulfate; Techniques; Technology; Therapeutic; Time; Tissues; Universities; Viral; base; depolymerization; design; experimental study; histological slides; improved; in vivo; innovation; mass spectrometer; phase 1 study; phase 2 study; quinoline; septic patients; success; tool

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.

该 SBIR 第一阶段提案旨在开发基于 LC-MS/MS 的 分析亚纳克范围内的硫酸乙酰肝素的方法。 高度硫酸化的多糖具有广泛的生物学功能。 对硫酸乙酰肝素定量和灵敏分析方法的强烈需求。 在此提案中，我们建议使用以下方法实现硫酸乙酰肝素的定量 为此，硫酸乙酰肝素被解聚成八种。 肝素裂解酶对二糖的组成进行分析。 二糖是通过 LC-MS/MS 方法完成的，这是我们的关键创新。 提出的方法是使用八个 13C 标记添加“绝对定量”功能 二糖校准物和一种 13C 标记的多糖校准物。 目标 1 旨在展示定量成分分析的能力 在第一阶段研究中，我们将重点关注来自小鼠组织和血浆的硫酸乙酰肝素。 分析健康小鼠和烧伤小鼠的血浆、肺、肝和肾中的 HS 老鼠。 目标 2 旨在对单一样品中的硫酸乙酰肝素进行定量分析 LC-MS/MS 方法的灵敏度将得到提高。 第一阶段研究，我们将分析来自肺部组织学切片的硫酸乙酰肝素， 来自健康和烧伤小鼠的肝脏和肾脏。 在 II 期研究中，我们计划将硫酸乙酰肝素的分析范围扩大到所有小鼠 我们还计划对健康小鼠和四只患病小鼠的组织进行分析。 来自 II 期研究中烧伤患者和脓毒症患者的临床样本。 该项目的成功将提供一种高灵敏度分析硫酸乙酰肝素的方法 生物学家的准确性和准确性，并将成为糖生物学研究的重大突破。