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(gly-can-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.
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 precolumn 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.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2010年第10期1992-1998,共7页
Chemical Journal of Chinese Universities
基金
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)
陕西省重点实验室重点科研计划项目(批准号:09JS081)资助