Isotopically Labeled Heparan Sulfate Glycosaminoglycan Disaccharides for use as Internal Standards

用作内标的同位素标记硫酸乙酰肝素糖胺聚糖二糖

基本信息

批准号：
10080563
负责人：
RON ORLANDO
金额：
$ 25.21万
依托单位：
GLYCOSCIENTIFIC, LLC
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10080563
关键词：
Acids Age Anabolism Biological Biology Carbohydrates Cell Line Cell surface Cells Cessation of life Chinese Hamster Ovary Cell Communities Complex DNA Detection Development Disaccharides Disease Enzymes Fluorescence GAG Gene Glucose Glucuronates Glucuronic Acids Glycosaminoglycans Goals Growth Health Heparin Heparitin Sulfate High Pressure Liquid Chromatography Human Hydrolysis Iduronic Acid Inflammation Process Investigation Isotope Labeling Label Libraries Life Light Lyase Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Measurable Measures Methods Modeling Monoclonal Antibodies Monosaccharides Names Nature Oligosaccharides Organism Parents Pathway interactions Patients Pharmacologic Substance Physiology Pilot Projects Polysaccharides Production Proteins RNA Recording of previous events Reference Standards Reporting Safety Secretory Vesicles Series Source Speed Structure Structure-Activity Relationship Techniques Testing Tissue Sample Tissues Uridine Diphosphate Sugars Vertebral column Work analog base developmental disease epimerase experimental study in situ imaging in vivo insight interest mast cell multiple reaction monitoring polysulfated glycosaminoglycan stable isotope therapeutic protein tool uptake virtual

Acids Age Anabolism Biological Biology Carbohydrates Cell Line Cell surface Cells Cessation of life Chinese Hamster Ovary Cell Communities Complex DNA Detection Development Disaccharides Disease Enzymes Fluorescence GAG Gene Glucose Glucuronates Glucuronic Acids Glycosaminoglycans Goals Growth Health Heparin Heparitin Sulfate High Pressure Liquid Chromatography Human Hydrolysis Iduronic Acid Inflammation Process Investigation Isotope Labeling Label Libraries Life Light Lyase Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Measurable Measures Methods Modeling Monoclonal Antibodies Monosaccharides Names Nature Oligosaccharides Organism Parents Pathway interactions Patients Pharmacologic Substance Physiology Pilot Projects Polysaccharides Production Proteins RNA Recording of previous events Reference Standards Reporting Safety Secretory Vesicles Series Source Speed Structure Structure-Activity Relationship Techniques Testing Tissue Sample Tissues Uridine Diphosphate Sugars Vertebral column Work analog base developmental disease epimerase experimental study in situ imaging in vivo insight interest mast cell multiple reaction monitoring polysulfated glycosaminoglycan stable isotope therapeutic protein tool uptake virtual

展开

项目摘要

PROJECT SUMMARY/ABSTRACT Sulfated glycosaminoglycan (GAG) carbohydrates represent one of the more structurally diverse groups of biomolecules, and a comprehensive understanding of their biological structure-function relationships has yet to be achieved. Unlike other biomolecules such as DNA/RNA and proteins that are synthesized based upon a template, GAG biosynthesis is the result of the cumulative actions of a series of enzymes to produce a dynamic, polydisperse mixture. The composition of this mixture is dependent on factors such as organism age, developmental or disease state and tissue of origin. Although this diversity presents a daunting analytical challenge, significant progress has been made in the field through attempts to isolate and characterize GAGs ranging from intact polysaccharides to enzymatically prepared oligosaccharides and disaccharides. The most widespread approach is to profile GAG disaccharides via separation (HPLC, UHPLC, HILIC, CE) and detection (UV, fluorescence, mass spectrometry (MS)). Domains can be characterized for structure- function studies by combining these techniques into hyphenated methods (e.g., LC-MS). Even though disaccharide analysis is the most widely utilized method for GAG analysis, there are no readily available sources of internal standards for MS-based quantitation. Recently, multiple reaction monitoring (MRM) has been increasingly applied to GAG analysis and internal standards would significantly enhance the quantitative nature of such approaches. We propose to leverage the biosynthetic machinery of CHO-S cells, which are widely employed in the production of protein pharmaceuticals and are known to produce GAGs, as a means to generate GAGs containing stable isotopes, currently named isoGAGs. Our approach will utilize 13C6 D-glucose and an in vivo method to introduce 15N into the UDP-sugar intermediates that form the backbone of the GAG chain. As these are stable isotopes, they do not add any safety concerns. Initial efforts will be focused on the creation of a series of heparan sulfate (HS) disaccharides that we envision as a commercially available library for quantitation during disaccharide profiling experiments.

项目摘要/摘要硫酸化的糖胺聚糖（GAG）碳水化合物代表了较为多样的群体之一生物分子，以及对其生物结构 - 功能关系的全面理解尚未是成就了。与其他基于模板合成的生物分子（例如DNA/RNA和蛋白质）不同，生物合成是一系列酶产生动态多分散混合物的累积作用的结果。该混合物的组成取决于生物年龄，发育或疾病状态和组织等因素起源。尽管这种多样性提出了艰巨的分析挑战，但在现场取得了重大进展通过试图隔离和表征从完整多糖到酶促制备的插孔寡糖和二糖。最广泛的方法是通过分离来介绍脱糖液（HPLC， UHPLC，HILIC，CE）和检测（UV，荧光，质谱法（MS））。可以将域特征在于结构 - 通过将这些技术结合到连字符方法（例如LC-MS）中来进行功能研究。即使二糖分析是用于插科打分析的最广泛使用的方法，没有随时可用的内部标准来源用于基于MS的定量。最近，多个反应监测（MRM）已越来越多地应用于GAG分析内部标准将显着提高此类方法的定量性质。我们建议利用 CHO-S细胞的生物合成机械，该机械广泛用于蛋白质药物的生产和已知会产生插科打,，作为生成含有稳定同位素（当前称为ISOGAGS的稳定同位素）的手段。我们的方法将利用13C6 D-葡萄糖和一种体内方法将15N引入形成的UDP-Sugar中间体中插科打链的骨干。由于这些是稳定的同位素，因此不会增加任何安全问题。最初的努力将是专注于创建一系列硫酸乙酰肝素（HS）二糖，我们将其视为商业上可用在二糖分析实验中进行定量的库。