基于棉花亲水色谱从鸡蛋黄中分离纯化唾液酸化糖肽研究  

Separation and Purification of Sialylglycopeptide from Egg Yolk Based on Cotton Hydrophilic Chromatography

作　　者：韩健利 陈庆辉 邹美义 路宇 魏明 李成 王承健 黄琳娟 王仲孚 HAN Jian-Li;CHEN Qing-Hui;ZOU Mei-Yi;LU Yu;WEI Ming;LI Cheng;WANG Cheng-Jian;HUANG Lin-Juan;WANG Zhong-Fu(Shaanxi Natural Carbohydrate Resource Engineering Research Center,College of Food Science and Technology,Northwest University,Xi'an 710069;College of Life Sciences,Northwest University,Xi'an 710069,China)

机构地区：[1]西北大学食品科学与工程学院陕西省天然多糖资源利用工程研究中心 [2]西北大学生命科学学院

出　　处：《分析化学》2020年第1期34-39,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.31670808,31870798)资助~~

摘　　要：鸡蛋黄中的唾液酸化糖肽(Sialylglycopeptide,SGP)的肽段部分由6个氨基酸(KVANKT)组成,其中天冬酰胺(N)被唾液酸化的复杂型N-糖链修饰。SGP主要从鸡蛋黄中分离获得,现有的SGP分离纯化方法操作繁琐,成本较高,难以规模化制备。本研究基于亲水相互作用色谱技术,用医用脱脂棉作为亲水色谱柱的固定相,建立了一种从鸡蛋黄中分离纯化唾液酸化糖肽的方法。首先将鸡蛋黄经苯酚处理得到唾液酸化糖肽的粗品,然后将糖肽粗品用水配成150 mg/mL的溶液,加入制备好的棉花亲水色谱柱中,依次用100%、95%、85%、75%(V/V)的乙腈-水溶液进行除杂,最后用去离子水洗脱唾液酸化糖肽。本方法操作简单,成本低廉。利用本方法从50个鸡蛋黄中制备得到了300 mg SGP,经高效液相色谱(HPLC)鉴定,其纯度高达95%,同时采用电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对SGP及其糖链组成和糖基化位点进行了表征。The peptide portion of the sialylglycopeptide(SGP)in egg yolk is composed of six amino acids residues(KVANKT),in which asparagine(N)is modified by sialylated complex N-glycan.SGP is mainly obtained from egg yolk,and currently,the methods for separation and purification of SGP are cumbersome,expensive,and difficult to scale production.In this study,a simple and low-cost method for separation and purification of sialylated glycopeptide from egg yolk was developed based on the hydrophilic interaction chromatography by using medical absorbent cotton as stationary phase.Firstly,50 egg yolks were treated with phenol to obtain crude sialylated glycopeptide.The obtained glycopeptide was then formulated into a 150 mg/mL solution and loaded onto the prepared cotton hydrophilic chromatographic column,followed by the treatment with 100%,95%,85%and 75%acetonitrile aqueous solution to remove the impurity.Finally,the sialyglycopeptide was obtained by eluting with deionized water.By this way,300 mg SGP was obtained and its purity was identified by high-performance liquid chromatography(HPLC)to be 95%.The glycan composition and glycosylation site of SGP were further validated by electrospray ionization mass spectrometry(ESI-MS)and tandem mass spectrometry(MS/MS).

关 键 词：唾液酸化糖肽 亲水色谱柱 脱脂棉 鸡蛋黄 

分 类 号：R28[医药卫生—中药学]