Development of analytical methods for glycosaminoglycans from biological sources

该SBIR第二阶段提案旨在开发一种基于LC-MS/MS的方法， 硫酸乙酰肝素和硫酸软骨素。硫酸乙酰肝素 和硫酸软骨素是高度硫酸化的多糖， 生物功能。对定量和灵敏的方法有强烈的需求 分析硫酸乙酰肝素和硫酸软骨素。在本提案中，我们建议 使用13 C标记的硫酸乙酰肝素和硫酸软骨素实现定量 校准品。为此，将硫酸乙酰肝素和硫酸软骨素解聚成 通过肝素裂解酶和软骨素ABCase。的组成分析 通过LC-MS/MS方法获得所得到的二糖。另外我们 计划使用13 C标记的硫酸乙酰肝素八糖校准品来测定 3-O-硫酸乙酰肝素水平。我们提出的方法的关键创新是 使用13 C标记的二糖校准品增加“绝对定量”能力， 13 C标记的八糖校准品。目标1旨在展示以下能力： 定量3-O-硫酸乙酰肝素的水平。我们将重点分析 血浆样品和人脑中3-O-硫酸化硫酸乙酰肝素的分析 患有神经退行性疾病的组织。目标2旨在开发一种方法， 硫酸软骨素的二糖定量分析。我们将合成六个13碳- 标记的硫酸软骨素双糖校准品进行研究。我们还将 进行人脑组织中硫酸软骨素的分析， 神经退行性疾病完成硫酸乙酰肝素和硫酸软骨素 血浆和实体组织的分析将证明该方法的耐用性。 该项目的成功将为硫酸乙酰肝素的分析提供一种方法， 硫酸软骨素的敏感性和准确性的生物学家。

This SBIR phase II proposal is aimed to develop a LC-MS/MS based method to analyze heparan sulfate and chondroitin sulfate with sub-nanogram quantities. Heparan sulfate and chondroitin sulfate are highly sulfated polysaccharides displaying a wide range of biological functions. There is a strong demand for a quantitative and sensitive method to analyze heparan sulfate and chondroitin sulfate. In this proposal, we propose to achieve the quantitation of heparan sulfate and chondroitin sulfate using 13C-labeled calibrants. To this end, heparan sulfate and chondroitin sulfate are depolymerized into disaccharides by heparin lyases and chondroitin ABCase. The composition analysis of the resultant disaccharides is accomplished by a LC-MS/MS method. In addition, we plan to use 13C-labeled heparan sulfate octasaccharide calibrants to determine the levels of 3-O-sulfated heparan sulfate. The crucial innovation in our proposed method is to add “absolute quantitation” capability using 13C-labeled disaccharide calibrants and 13C-labeled octasaccharide calibrants. Aim 1 is designed to demonstrate the ability to quantitative the level of 3-O-sulfated heparan sulfate. We will focus on analyzing the analysis of 3-O-suflated heparan sulfate from plasma samples and from human brain tissues with neurodegenerative diseases. Aim 2 is aimed to develop a method for quantitative disaccharide analysis of chondroitin sulfate. We will synthesize six 13C- labeled chondroitin sulfate disaccharide calibrants to carry out the studies. We will also conduct the analysis for chondroitin sulfate from human brain tissues from neurodegenerative diseases. The completed heparan sulfate and chondroitin sulfate analysis from plasma and solid tissues will demonstrate the robustness of the method. The success of this project will provide a method to analyze heparan sulfate and chondroitin sulfate with sensitivity and accuracy for biologists.