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 方法分析亚纳克级硫酸乙酰肝素。硫酸乙酰肝素是一种 高度硫酸盐化的多糖具有广泛的生物学功能。的确有 迫切需要一种定量和灵敏的方法来分析硫酸乙酰肝素。在……里面 在这项建议中，我们建议使用 双糖分析。为此，硫酸乙酰肝素被解聚为八个 通过肝素裂解酶合成二糖。合成产物的成分分析 二糖采用LC-MS/MS联用的方法进行分析。我们的关键创新是 提出的方法是使用八个13C标记的方法来增加“绝对定量”能力 二糖标定剂和一种13C标记的多糖标定剂。 目标1旨在演示定量成分分析的能力 小鼠组织和血浆中的硫酸乙酰肝素。在第一阶段的研究中，我们将集中于 健康及烧伤小鼠血浆、肺、肝、肾组织中HS的分析 老鼠。 目的2目的是对单一来源的硫酸肝素进行定量分析。 组织学切片。LC-MS/MS方法的灵敏度将得到提高。在 第一阶段研究，我们将分析肺组织切片中的硫酸乙酰肝素， 来自健康和烧伤小鼠的肝脏和肾脏。 在第二阶段的研究中，我们计划将硫酸肝素的分析扩大到所有小鼠 来自健康小鼠和四只患病小鼠的组织。我们还计划进行分析 来自II期研究的烧伤患者和脓毒症患者的临床样本。这个 该项目的成功将为硫酸乙酰肝素的灵敏分析提供一种方法 并将成为糖生物学研究的一个重大突破。