Pronase E Digestion of N-Glycans in Glycoprotein and Its Fluorescent Derivatives Analysis by HPLC-ESI/MS

Chem. J. Chinese Universities ›› 2010, Vol. 31 ›› Issue (10): 1992.

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Pronase E Digestion of N-Glycans in Glycoprotein and Its Fluorescent Derivatives Analysis by HPLC-ESI/MS

XU Sha1, ZHANG Ping1,2, HUANG Lin-Juan1, WANG Zhong-Fu1*

1. Key Laboratory of Ministry of Education for Western China Resource Biology and Biotechnology, College of Life Sciences, Northwest University, Xi′an 710069, China;
2. Chemistry and Chemical Engineering School, Xianyang Normal University, Xianyang 712000, China

Received:2009-12-28 Online:2010-10-10 Published:2010-10-10
Contact: WANG Zhong-Fu. E-mail: wangzhf@nwu.edu.cn
Supported by:
国家“八六三”计划项目(批准号: 2007AA10Z338, 2007AA091601)、教育部新世纪优秀人才支持计划(批准号: NCET-08-0893)和陕西省重点实验室重点科研计划项目(批准号: 09JS081)资助.

Abstract

Abstract: A method for non-specific enzymatic digestion of glycopeptides to release N-glycans from glycoprotein was developed. Pronase E instead of the traditional PNGase F was used to release glycopeptides from Ribo B and Chicken Albumin, and under the optimized digestion conditions that the quality ratio of the Pronase E to glycoprotein was 1∶1, the closed-ring oligosaccharides named glycans-Asn with a single amino acid(Asn) were obtained. A modified 9-fluorenylmethyl chloroformate(Fmoc-Cl) pre-column derivatization procedure was also applied to these glycans-Asn. And the Fmoc-Cl labeled glycans-Asn products were characterized by high performance liquid chromatography/electrospray ionization mass spectrometry(HPLC-ESI/MS). So a new method to digest glycoprotein with Pronase E and analyze trace glycans from glycoprotein with Fmoc-Cl pre-column derivatization by HPLC-ESI/MS is established. This method not only remains the native structure of N-glycans, but also provides possibility of fluorescent derivation with —NH2 function groups, which brings the convenience of separation and preparation of N-glycans and study the interaction between glycan and proteins.

Key words: Pronase E, Glycoprotein, 9-Fluorenylmethyl chloroformate, ESI-MS, Fluorescent derivative

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XU Sha, ZHANG Ping, HUANG Lin-Juan, WANG Zhong-Fu*. Pronase E Digestion of N-Glycans in Glycoprotein and Its Fluorescent Derivatives Analysis by HPLC-ESI/MS[J]. Chem. J. Chinese Universities, 2010, 31(10): 1992.

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Pronase E Digestion of N-Glycans in Glycoprotein and Its Fluorescent Derivatives Analysis by HPLC-ESI/MS

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