Comparison of Methods to Release Mucin-Type O‑Glycans for Glycomic Analysis
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...
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Published in | Analytical chemistry (Washington) Vol. 89; no. 17; pp. 8870 - 8876 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
05.09.2017
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/acs.analchem.7b01346 |