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Updated: Jan 26, 2026

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Published on: April 27, 2010
A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase
Akihiko Kameyama1, Wai Wai Thet Tin1, Masaaki Toyoda2
1Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umesono, Tsukuba, Ibaraki, 305-8568, Japan.
Abstract:
O-Linked glycan liberation from proteins through reductive beta-elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-linked glycan analyses.
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