title: Improved nonreductive O-glycan release by hydrazinolysis with ethylenediaminetetraacetic acid addition.
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.
authors.affiliation: Ludger, Culham Science Centre, Oxfordshire OX14 3EB, UK. Electronic address: rad.kozak@ludger.com.
authors.forename: Radoslaw P
authors.initials: RP
authors.lastname: Kozak
authors.affiliation: Ludger, Culham Science Centre, Oxfordshire OX14 3EB, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Royle
authors.affiliation: Ludger, Culham Science Centre, Oxfordshire OX14 3EB, UK.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gardner
authors.affiliation: Department of Rheumatology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands; Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands.
authors.forename: Albert
authors.initials: A
authors.lastname: Bondt
authors.affiliation: Ludger, Culham Science Centre, Oxfordshire OX14 3EB, UK.
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Fernandes
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands; Division of Bioanalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D006834:Hydrazines; D011134:Polysaccharides; D004492:Edetic Acid
keywords: Glycan release; HILIC–HPLC; LC–ion trap–MS/MS; O-linked glycans; Peeling