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Separation and preparation of N-glycans based on ammonia-catalyzed release method

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Abstract

The study of carbohydrates requires large amounts of glycans. N-Glycans can be synthesized but generating large quantities of N-glycans with diverse structures remains difficult. In this study, we aimed to obtain large amounts of glycans using an optimized procedure. Two types of reductive N-glycans were released from chicken egg albumin (ovalbumin) and soy protein using an ammonia catalysis method and labeled with benzenesulfonyl hydrazide (BSH). After preliminary separation by preparative HPLC, N-glycan-BSH components were de-labeled separately and reducing N-glycans were recovered. The de-labeled reducing N-glycans were derived with different labeling reagents and further separated and purified with two/multi-dimensional HPLC for various studies. We selected the bifunctional reagent 2-amino-N-(2-aminoethyl)-benzamide (AEAB) as a labeling reagent combined with C18 column for two-dimensional HPLC separation. A total of 21 and 8 N-glycan-AEAB conjugates were obtained from ovalbumin and soy protein, respectively. A reactive primary alkylamine of N-glycan-AEAB conjugates can be effectively immobilized on microarray surfaces, allowing for subsequent functional studies of glycans.

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Acknowledgments

This work was supported by the National Natural Science Fundation of China (grant nos. 31670808, 31870798) and National Key Research and Development Program of China (grant no. 2018YFD0901101).

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Author notes

  1. Meifang Yang and Ming Wei contributed equally to this work.

Authors and Affiliations

  1. The College of Life Sciences, Northwest University, Xi’an, 710069, China

    Meifang Yang, Ming Wei, Chengjian Wang, Yu Lu, Wanjun Jin, Xi Gao, Cheng Li, Linjuan Huang & Zhongfu Wang

  2. Glycobiology and Glycotechnology Research center, College of Food Science and Technology, Northwest University, Xi’an, 710069, China

    Chengjian Wang, Langhong Wang, Linjuan Huang & Zhongfu Wang

Authors
  1. Meifang Yang
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  2. Ming Wei
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Correspondence to Linjuan Huang or Zhongfu Wang.

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Yang, M., Wei, M., Wang, C. et al. Separation and preparation of N-glycans based on ammonia-catalyzed release method. Glycoconj J 37, 165–174 (2020). https://doi.org/10.1007/s10719-020-09909-z

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  • DOI: https://doi.org/10.1007/s10719-020-09909-z

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