液质联用技术分析可溶性CD95-Fc融合蛋白N-糖组成  

N-glycan analysis of soluble CD95-Fc by UPLC/MS

作　　者：于雷[1] 陶磊[1] 李响[1] 郭莹[1] 饶春明[1] YU Lei;TAO Lei;LI Xiang;GUO Ying;RAO Chun-ming(National Institute of Food and Drug Control, Beijing 100050, China)

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国新药杂志》2018年第13期1496-1501,共6页Chinese Journal of New Drugs

基　　金：国家"重大新药创制"科技重大专项资助项目(2015ZX09501008-001);中国食品药品检定研究院中青年发展研究基金资助项目(2017B3)

摘　　要：目的:分析鉴定可溶性CD95-Fc融合蛋白的N-糖组成。方法:采用Glyco Works Rapi Fluor-MS N-糖分析试剂盒酶解并标记、纯化N-糖基,糖基纯化产物经Waters BEH Glycan糖蛋白分析柱分离后,依次进入荧光检测器和飞行时间质谱仪检测。结果:结合荧光和质谱检测结果,鉴定出15种糖型:G0-GN,G0FGN,G0,G0F,Man5,G1F,G1F,G2F,G2F+SA,G2F+SA,G3F,G2F+2SA,G3F+SA,G3F+2SA和G3F+3SA;各糖型组分实测单同位素分子量与理论值差异均不足0.1Da;所占比例最高的糖型依次为G0F(30%),G1F(25%),G2F+SA(14%)和G2F(13%);不同批次相同糖基组分保留时间的RSD均低于0.2%,峰面积百分比的RSD均低于15%。结论:本研究成功鉴定出可溶性CD95-Fc融合蛋白的15种主要糖型,不同批次的糖型荧光图谱基本一致,可作为UPLC法对可溶性CD95-Fc融合蛋白N-糖定性定量的标准参考图谱。Objective： To analyze and identify N-glycans of soluble CD95-Fc. Methods： Digest,label and purify N-glycans by Glyco Works Rapi Fluor Fluor-MS N-Glycan Kit,and the purified N-glycans were separated by Waters BEH Glycan column,and detected by fluorescence detector and Q-Tof MS. Results： Combined the results of fluorescence detector and MS,15 glycans were identified： G0-GN,G0 F-GN,G0,G0 F,Man5,G1 F,G1 F,G2 F,G2 F ＋ SA,G2 F ＋ SA,G3 F,G2 F ＋ 2 SA,G3 F ＋ SA,G3 F ＋ 2 SA and G3 F ＋ 3 SA. The difference of measured and theoretical monoisotoplic MW of individual glycan was below 0. 1 Da. The glycans with the highest percentage were G0 F（ - 30%）,G1 F（ - 25%）,G2 F ＋ SA（ - 14%） and G2 F（ - 13%）. The RSDs of retention time among different batches were below 0. 2%,and the RSDs of area percentage were below 15%. Conclusions：This study successfully identified 15 N-glycans of soluble CD95-Fc,and the fluorescence spectra of different batches were roughly identical,which could be standard reference spectrum for qualitative and quantitative analysis of soluble CD95-Fc by UPLC.

关 键 词：可溶性CD95-Fc N-糖分析 超高效液相/质谱 荧光糖谱 糖型鉴定 

分 类 号：R917[医药卫生—药物分析学]