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The ammonia-catalyzed release of glycoprotein N-glycans

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Abstract

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.

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Acknowledgements

This work was supported by the National Natural Scince Foundation of China (31670808, 31600647, 31370804, 21375103), the Natural Science Special Fund of Shaanxi Provincial Education Department (16JK1782), the Scientific Research Program Fund for Shaanxi Province Key laboratory (16JS109) and the Scientific Research Foundation of Northwest University, China (15NW18).

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  1. Chengjian Wang and Meifang Yang contributed equally to this work.

Authors and Affiliations

  1. The Education Ministry Key Laboratory of Resource Biology and Biotechnology in Western China and Shaanxi Provincial Key Laboratory of Biotechnology, College of Life Sciences, Northwest University, No. 229 North Taibai Road, Xi’an, 710069, Shaanxi Province, China

    Chengjian Wang, Meifang Yang, Xi Gao, Cheng Li, Zihua Zou, Jianli Han, Linjuan Huang & Zhongfu Wang

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  1. Chengjian Wang
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  2. Meifang Yang
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Correspondence to Linjuan Huang or Zhongfu Wang.

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Wang, C., Yang, M., Gao, X. et al. The ammonia-catalyzed release of glycoprotein N-glycans. Glycoconj J 35, 411–420 (2018). https://doi.org/10.1007/s10719-018-9827-6

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