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

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.

项目概要/摘要硫酸化糖胺聚糖 (GAG) 碳水化合物是结构更加多样化的一类碳水化合物。生物分子，以及对其生物结构与功能关系的全面了解实现了。与其他生物分子（例如 DNA/RNA 和基于模板合成的蛋白质）不同，GAG 生物合成是一系列酶累积作用的结果，产生动态的多分散混合物。该混合物的成分取决于生物体年龄、发育或疾病状态和组织等因素原产地。尽管这种多样性带来了艰巨的分析挑战，但该领域已经取得了重大进展通过尝试分离和表征从完整多糖到酶法制备的 GAG 寡糖和二糖。最广泛的方法是通过分离（HPLC、 UHPLC、HILIC、CE）和检测（UV、荧光、质谱 (MS)）。域可以表征结构- 通过将这些技术结合到联用方法（例如 LC-MS）中进行功能研究。尽管二糖分析是最广泛使用的 GAG 分析方法，但没有现成的内标来源用于基于 MS 的定量。近年来，多反应监测（MRM）越来越多地应用于GAG分析内部标准将显着增强此类方法的定量性质。我们建议利用 CHO-S细胞的生物合成机制，广泛应用于蛋白质药物的生产和已知可以产生 GAG，作为产生含有稳定同位素的 GAG（目前称为 isoGAG）的一种手段。我们的该方法将利用 13C6 D-葡萄糖和体内方法将 15N 引入 UDP-糖中间体中，形成 GAG链的主干。由于这些是稳定同位素，因此不会增加任何安全问题。初步努力将是专注于创建一系列硫酸乙酰肝素 (HS) 二糖，我们设想将其作为商业化产品用于二糖分析实验期间定量的文库。