Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

高等学校化学学报 ›› 2010, Vol. 31 ›› Issue (10): 1992.

Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析

徐莎1, 张萍1,2, 黄琳娟1, 王仲孚1

1.西北大学生命科学学院西部资源生物与现代生物技术教育部重点实验室, 西安 710069;
2. 咸阳师范学院化学与化工学院, 咸阳 712000

收稿日期:2009-12-28 出版日期:2010-10-10 发布日期:2010-10-10
通讯作者: 王仲孚, 男, 博士, 教授, 博士生导师, 主要从事糖生物学与糖工程学研究. E-mail: wangzhf@nwu.edu.cn
基金资助:
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.

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.

摘要/Abstract

摘要： 建立了一种用非特异性酶链酶蛋白酶 E(Pronase E)从糖蛋白上释放N-糖链的方法. 以牛胰核糖核酸酶 B(Ribo B)和鸡白蛋白(Chicken Albumin)为材料, 用Pronase E代替N-糖苷酶 F(PNGase F)释放N-糖链. 当蛋白酶质量与糖蛋白质量比为1∶1时, 得到只带一个天冬氨酸(Asn)的闭环N-糖链, 称其为糖氨酸(glycan-Asn), 这样既为糖链引入了天然的—NH2活性基团, 同时还保持了糖链原有的还原端闭环结构. 以9-氯甲酸芴甲酯(Fmoc-Cl)为衍生试剂对解离后的糖氨酸进行衍生, 采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI/MS)对Fmoc-Cl糖氨酸衍生物进行分析, 建立了糖蛋白的Pronase E酶解、微量糖氨酸的Fmoc-Cl衍生以及糖氨酸衍生物的HPLC-ESI/MS分析方法, 该方法保持了N-糖链的天然结构, 便于以—NH2为功能基团进一步进行荧光标记、分离制备以及糖链与蛋白质的相互作用研究.

关键词: Pronase E, 糖蛋白, 9-氯甲酸芴甲酯, 电喷雾质谱, 荧光标记

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

TrendMD:

徐莎, 张萍, 黄琳娟, 王仲孚. Pronase E酶解释放糖蛋白N-糖链的方法及荧光标记衍生物的LC-MS分析. 高等学校化学学报, 2010, 31(10): 1992.

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

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