Comparative Quantification Method for Glycosylated Products Elongated on β-Xylosides Using a Stable Isotope-Labeled Saccharide Primer
Comparative Quantification Method for Glycosylated Products Elongated on β-Xylosides Using a Stable Isotope-Labeled Saccharide Primer
复制标题
使用稳定同位素标记的糖引物对 β-木糖苷延伸的糖基化产物进行比较定量方法
DOI：
10.1021/acs.analchem.7b05438
复制
发表时间：
2018
期刊：
影响因子：
7.4
通讯作者：
Toshinori Sato
中科院分区：
文献类型：
--
作者：
Yuya Otsuka;Toshinori Sato
细胞产生的糖胺聚糖(GAG)的结构和数量引起了人们的极大兴趣，因为GAG的生物合成活性可以在疾病和分化等细胞过程中发生变化。β-木糖苷，又称糖引子，已被用作人工受体，不仅用于在细胞和组织中产生Gag寡糖，而且还用于研究它们的生物合成途径。已有多种分析方法被应用于确定使用糖引物法延伸的GAG寡糖的结构和数量，但分析中的固相萃取等样品制备过程会造成实验误差，并扰乱糖基化产物的准确比较定量。在这项研究中，我们开发了一种新的定量方法，使用氚标记的糖引物。化学合成了“重”和“轻”两个引物，并用液相色谱-串联质谱仪对其引发能力进行了确认。轻、重质产物的相对峰面积呈良好的线性关系，与糖基化产物的理论量有很好的相关性。然后，作为一项验证研究，我们使用已知的GAG生物合成抑制剂进行了生物合成抑制试验。根据糖引物法的相对定量，四种GAG生物合成抑制剂的作用模式的差异取决于GAG生物合成途径。我们的结果表明，该方法可能会为利用培养的细胞和组织进行糖胺糖凝胶的比较开辟一条新的途径。
The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. β-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The “heavy” and “light” primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.