The Laboratory of Toxicology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka 584-8540, Japan.
Institute for Glyco-core Research (iGCORE), Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
J Proteome Res. 2024 Oct 4;23(10):4254-4272. doi: 10.1021/acs.jproteome.4c00240. Epub 2024 Sep 10.
Minor acidic glycans, such as sulfated and phosphorylated glycans, constitute only a small fraction of biological glycome, making their analysis a considerable challenge. In this study, we developed a technique to analyze minor acidic -glycans in biological samples. First, efficient reaction conditions for the release of -glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH spin columns. The performance of the established method was evaluated using mucin samples, and sulfated -glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic -glycans in cultured cancer cells. In addition to trifucosylated sulfated -glycans and disulfated -glycans, sulfated -glycans with KDN were detected in LS174T cells. The relative amount of sulfated glycans in LS174T cells was almost 10-fold higher than that in the other cells. Moreover, a large polylactosamine-type sulfated -glycan with a molecular weight >3500 was detected in MKN45 cells. Interestingly, phosphorylated ribose, possibly bound to serine/threonine, was observed in all the cells used in this study. Thus, our established analytical method allows for the analysis of minor acidic -glycans that cannot be detected using existing glycomics methods.
微量酸性糖，如硫酸化和磷酸化糖，仅占生物糖组的一小部分，因此对其进行分析是一项艰巨的挑战。在这项研究中，我们开发了一种分析生物样品中微量酸性糖的技术。首先，确定了从蛋白质中释放糖的有效反应条件。接下来，使用 NH 旋转柱建立了一种用于回收微量酸性糖的高通量方法。使用粘蛋白样品评估了所建立方法的性能，并成功检测到牛颌下腺粘蛋白和猪胃粘蛋白中的硫酸化糖。我们还分析了培养的癌细胞中的微量酸性糖。除了三岩藻糖基硫酸化糖和二硫酸化糖外，LS174T 细胞中还检测到带有 KDN 的硫酸化糖。LS174T 细胞中硫酸化糖的相对含量几乎是其他细胞的 10 倍。此外，在 MKN45 细胞中还检测到一种分子量>3500 的大聚乳糖胺型硫酸化糖。有趣的是，在本研究中使用的所有细胞中都观察到了可能与丝氨酸/苏氨酸结合的磷酸化核糖。因此，我们建立的分析方法允许分析使用现有糖组学方法无法检测到的微量酸性糖。
