Comparison of Methods to Release Mucin-Type O‑Glycans for Glycomic Analysis

• Published in: Analytical chemistry (Washington) Vol. 89; no. 17; pp. 8870 - 8876
• Main Authors: Goso, Yukinobu, Sugaya, Tsukiko, Ishihara, Kazuhiko, Kurihara, Makoto
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.09.2017
• Subjects: Ammonia; Analytical chemistry; Anthranilic acid; Chemistry; Chromatography; Fluorescence; Glycan; Glycoproteins; High performance liquid chromatography; Hydrazine; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Mucin; Polysaccharides; Proteins; Rodents; Sodium; Sodium hydroxide
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.7b01346

Mucin-type O-glycans (O-glycans) are one of the most common glycans attached to proteins. To develop an optimized glycomic analysis protocol, O-glycans were released from glycoproteins using hydrazine, ammonia, or sodium hydroxide treatment, followed by hydrophilic interaction liquid chromatography to evaluate O-glycan release. We found that porcine gastric mucin or bovine fetuin treated at 60 °C for 6 h with hydrazine gas in the presence of malonic acid yielded O-glycans with only a small amount of degraded, so-called “peeled” products. Ammonia treatment also yielded intact O-glycans but with additional peeled products containing GlcNAc at the reducing end. In contrast, sodium hydroxide treatment yielded mainly peeled glycans, including those containing GlcNAc at the reducing end. Importantly, O-glycans obtained from rat gastric mucin treated with hydrazine and labeled with anthranilic acid had a nearly identical profile following hydrophilic interaction liquid chromatography as permethylated O-glycan alditols analyzed by mass spectroscopy. Taken together, the data suggest that glycan release using hydrazine treatment, followed by high-performance liquid chromatography after fluorescent labeling, is a suitable method for glycomic analysis of mucin-type O-glycans.