Isotopically Labeled Heparan Sulfate Glycosaminoglycan Disaccharides for use as Internal Standards

项目摘要/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 生物分子，以及对它们的生物结构-功能关系的全面了解还有待于 已实现。与基于模板合成的DNA/RNA和蛋白质等其他生物分子不同，Gag 生物合成是一系列酶的累积作用的结果，以产生动态的、多分散的混合物。 这种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 原产地。尽管这种多样性是一个艰巨的分析挑战，但在这一领域已经取得了重大进展。 通过尝试分离和鉴定从完整的多糖到酶法制备的GAG 低聚糖和二糖。最广泛的方法是通过分离(高效液相色谱， UHPLC、HILIC、CE)和检测(UV、荧光、质谱仪)。域可以被表征为结构- 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。即使是双糖 分析是使用最广泛的GAG分析方法，没有现成的内标来源 用于基于MS的定量。近年来，多反应监测技术(MRM)越来越多地应用于Gag的分析 而内部标准将大大提高这种方法的数量性质。我们建议利用 广泛应用于蛋白质药物和蛋白质药物生产的CHO-S细胞的生物合成机制 已知产生GAG，作为产生含有稳定同位素的GAG的一种手段，目前称为等GAGs。我们的 该方法将利用13C6 D-葡萄糖和体内方法将15N引入形成的UDP-糖中间体 恶作剧链的中坚力量。由于这些是稳定的同位素，它们不会增加任何安全问题。初步努力将是 专注于创造一系列的硫酸乙酰肝素(HS)双糖，我们设想这是一种商业上可用的 双糖图谱实验中的定量文库。

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.