Systems Biology and Medicine Center for Complex Diseases, Affiliated Hospital of Qingdao University, Qingdao, China.
Methods Mol Biol. 2022;2303:297-306. doi: 10.1007/978-1-0716-1398-6_24.
Glycosaminoglycans (GAGs) are heterogeneous biomacromolecules made by all animal cells with overlapping molecular weight and high negative charge densities, which make thorough separation of different types of GAGs and elimination of all GAG-binding proteins difficult. Even with the constant challenge of quality control, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin glycosaminoglycans (GAGs) have been used as nutraceuticals and modern drugs for many years worldwide. Testing galactosamine in heparin has been added to the USP monograph after contaminated heparin event, but the general monosaccharide composition analysis has not been developed for GAG quality control purposes. Using a PCR-facilitated hydrolysis assay, the hydrolyzed GAG saccharides were labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) and quantified by high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Glucosamine was found in both chondroitin sulfate and dermatan sulfate whereas galactosamine was observed in both heparan sulfate and heparin, indicating the cross contamination among different types of GAGs. Moreover, fucose was detected in chondroitin sulfate, dermatan sulfate, and heparan sulfate, and both fucose and mannose were detected in chondroitin sulfate, suggesting the co-presence of other types of glycans or novel fucosylated GAG structures. Furthermore, both the amount and structure of acid-resistant disaccharides provide distinguishable features for each type of GAGs at the same hydrolysis condition. Thus, monosaccharide analysis provides a practical and quantitative way for GAG quality control.
糖胺聚糖（GAGs）是由所有动物细胞产生的异质生物大分子，分子量重叠且带高度负电荷密度，这使得不同类型的 GAGs 的彻底分离和所有 GAG 结合蛋白的去除变得困难。尽管一直面临质量控制的挑战，但硫酸软骨素、硫酸皮肤素、硫酸乙酰肝素和肝素糖胺聚糖（GAGs）多年来已在全球范围内作为营养保健品和现代药物使用。在污染肝素事件后，USP 专论中已添加了肝素中半乳糖胺的检测，但尚未为 GAG 质量控制目的开发出一般单糖组成分析。本研究采用聚合酶链反应（PCR）辅助水解测定法，用 1-苯基-3-甲基-5-吡唑啉酮（PMP）标记水解的 GAG 糖，并通过高效液相色谱（HPLC）与质谱（MS）联用进行定量。在硫酸软骨素和硫酸皮肤素中发现了葡萄糖胺，而在硫酸乙酰肝素和肝素中发现了半乳糖胺，表明不同类型的 GAG 之间存在交叉污染。此外，在硫酸软骨素、硫酸皮肤素和硫酸乙酰肝素中检测到岩藻糖，在硫酸软骨素中同时检测到岩藻糖和甘露糖，表明存在其他类型的聚糖或新型岩藻糖基 GAG 结构。此外，在相同的水解条件下，耐酸二糖的数量和结构为每种 GAG 提供了可区分的特征。因此，单糖分析为 GAG 质量控制提供了一种实用且定量的方法。
