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

• 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
• 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

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.

项目总结/摘要 硫酸化糖胺聚糖（GAG）碳水化合物代表了结构上更多样化的糖胺聚糖组之一。 生物分子，并且对其生物结构-功能关系的全面理解还有待于 办妥了一批与基于模板合成的其他生物分子如DNA/RNA和蛋白质不同，GAG 生物合成是一系列酶累积作用的结果，以产生动态的多分散混合物。 该混合物的组成取决于诸如生物体年龄、发育或疾病状态和组织等因素。 混元虽然这种多样性给分析工作带来了巨大挑战，但在这一领域已取得了重大进展 通过尝试分离和表征从完整多糖到酶促制备的GAG， 寡糖和二糖。最普遍的方法是通过分离（HPLC， UHPLC、HILIC、CE）和检测（UV、荧光、质谱（MS））。结构域可以通过结构来表征- 通过将这些技术结合到连字符方法中进行功能研究（例如，LC-MS）。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）已越来越多地应用于GAG分析 和内部标准将大大加强这种办法的数量性质。我们建议利用 CHO-S细胞的生物合成机制，其广泛用于蛋白质药物的生产， 已知产生GAG，作为产生含有稳定同位素的GAG（目前称为isoGAG）的手段。我们 这种方法将利用13C6 D-葡萄糖和体内方法将15N引入UDP-糖中间体， GAG链的主干由于它们是稳定的同位素，它们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，我们设想作为一种商业上可用的 在二糖分析实验期间用于定量的文库。