Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea.
Department of Biology, Changwon National University, Changwon, 51140, Republic of Korea.
Anal Biochem. 2021 May 15;621:114152. doi: 10.1016/j.ab.2021.114152. Epub 2021 Mar 13.
Changes in glycan levels could directly affect the biochemical properties of glycoproteins and thus influence their physiological functions. In order to decode the correlation of glycan prevalence with their physiological contribution, many mass spectrometry (MS) and stable isotope labeling-based methods have been developed for the relative quantification of glycans. In this study, we expand the quantitative glycomic toolbox with the addition of optimized Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) approach in baker's yeast (Saccharomyces cerevisiae). We demonstrate that culturing baker's yeast in the presence of carbon-13 labeled glucose (1-C) leads to effective incorporation of carbon-13 to both N-linked and O-linked glycans. We established that metabolic incorporation of isotope-labeled glucose at a concentration of 5 mg/mL for three days is required for an accurate quantitative analysis with optimal isotopic cluster distribution of glycans. To validate the robustness of the method, we performed the analysis by 1:1 mixing of normal and isotope-labeled glycans, and obtained excellent linear calibration curves from various analytes. Finally, we quantitated the inhibitory effect of tunicamycin, a N-linked glycosylation inhibitor, to glycan expression profile in yeast.
聚糖水平的变化可能直接影响糖蛋白的生化特性，从而影响其生理功能。为了解码聚糖流行与生理贡献之间的相关性，已经开发了许多基于质谱 (MS) 和稳定同位素标记的方法来相对定量聚糖。在这项研究中，我们通过在面包酵母 (Saccharomyces cerevisiae) 中添加经过优化的代谢同位素标记多糖的方法 (MILPIG)，扩展了定量糖组学工具包。我们证明，在存在 13C 标记葡萄糖 (1-C) 的情况下培养面包酵母，可有效将 13C 掺入到 N 连接和 O 连接的聚糖中。我们确定，在 5mg/mL 的浓度下代谢掺入同位素标记的葡萄糖三天，对于具有最佳糖基化异构体分布的准确定量分析是必需的。为了验证该方法的稳健性，我们通过正常和同位素标记的聚糖 1:1 混合进行了分析，并且从各种分析物中获得了极好的线性校准曲线。最后，我们定量测定了 N-连接糖基化抑制剂衣霉素对酵母中糖蛋白表达谱的抑制作用。
