Developing Method for Minor Acidic <i>O</i>-Glycan Analysis in Mucin and Cancer Cell Samples

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 <i>O</i>-glycans in biological samples. First, efficient reaction conditions for the release of <i>O</i>-glycans from the proteins were determined. Next, a high-throughput method was established for the recovery of minor acidic glycans using NH₂ spin columns. The performance of the established method was evaluated using mucin samples, and sulfated <i>O</i>-glycans were successfully detected in bovine submaxillary gland mucin and porcine stomach mucin. We also analyzed the minor acidic <i>O</i>-glycans in cultured cancer cells. In addition to trifucosylated sulfated <i>O</i>-glycans and disulfated <i>O</i>-glycans, sulfated <i>O</i>-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 <i>O</i>-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 <i>O</i>-glycans that cannot be detected using existing glycomics methods.