Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition |
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Authors: | Radoslaw P Kozak Louise Royle Richard A Gardner Albert Bondt Daryl L Fernandes Manfred Wuhrer |
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Institution: | 1. Ludger, Culham Science Centre, Oxfordshire OX14 3EB, UK;2. Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands;3. Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands;4. Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands |
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Abstract: | The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as “peeling” often compromises hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release. |
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Keywords: | O-linked glycans Glycan release Peeling HILIC&ndash HPLC LC&ndash ion trap&ndash MS/MS |
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