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的分析方法 亚纳克量的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盐化的多糖，显示出广泛的 生物功能。对一种定量和灵敏的方法有强烈的需求 对硫酸乙酰肝素和硫酸软骨素进行分析。在这项建议中，我们建议 用~(13)C标记实现硫酸肝素和硫酸软骨素的定量 校准器。为此，硫酸乙酰肝素和硫酸软骨素解聚为 二糖由肝素裂解酶和软骨素ABCase组成。关于化学成分分析 通过LC-MS/MS方法完成了双糖的合成。此外，我们 计划使用13C标记的硫酸乙酰肝素八糖校准剂来确定 3-O-硫酸乙酰肝素的水平。我们提出的方法的关键创新是 使用13C标记的双糖校准剂和 13C标记的八糖标定剂。AIM 1旨在展示 定量测定3-O-硫酸乙酰肝素的含量。我们将重点分析 血浆和人脑组织中3-O-硫代乙酰肝素的分析 神经退行性疾病的组织。目标2旨在开发一种方法 硫酸软骨素双糖定量分析。我们将合成六个13C- 标记的硫酸软骨素二糖校准剂进行研究。我们还将 从人脑组织中分析硫酸软骨素 神经退行性疾病。完全硫酸肝素和硫酸软骨素 对血浆和固体组织的分析将证明该方法的稳健性。 该项目的成功将提供一种分析硫酸乙酰肝素和 对于生物学家来说，硫酸软骨素具有敏感度和准确性。

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.