多种质谱解离技术对缩宫素结构表征的比较研究  

Characterization of Oxytocin by Different Kinds of Dissociation Methods Using High Resolution Mass Spectrometry:a Comparative Study

作　　者：钱建钦 何佳佳 朱坤丹 梁键谋 黄巧巧 QIAN Jianqin;HE Jiajia;ZHU Kundan;LIANG Jianmou;HUANG Qiaoqiao(Zhejiang Institute for Food and Drug Control,Key Laboratory of Core Technology for Generic Drug Evaluation,National Medical Products Administration,Hangzhou 310052,China)

机构地区：[1]浙江省食品药品检验研究院,国家药品监督管理局仿制药评价关键技术重点实验室,杭州310052

出　　处：《中国现代应用药学》2023年第6期803-807,共5页Chinese Journal of Modern Applied Pharmacy

基　　金：浙江省食品药品监督管理局科技计划项目(2019008)。

摘　　要：目的采用静电场轨道阱高分辨质谱仪,比较4种质谱解离技术在缩宫素多肽结构表征方面的应用。方法采用内层涂布C_(18)填料的毛细管色谱柱,以0.1%甲酸为流动相A,0.1%甲酸-乙腈(20∶80)为流动相B,梯度洗脱。电喷雾离子源正离子模式下,采用碰撞诱导裂解(collision induced dissociation,CID)、高能C阱碰撞解离(higher-energy collision dissociation,HCD)、电子转移解离(electron transfer dissociation,ETD)、电子转移/高能碰撞诱导解离(electron transfer/high energy collision dissociation,EThcD)4种解离技术,采集高分辨二级质谱图,比较不同解离技术在氨基酸序列表征方面的异同。结果EThcD产生的特征性碎片离子最多,对氨基酸序列覆盖度最高,其次为ETD、HCD,而CID的覆盖度相对较低,且CID在缩宫素分子内二硫键的表征方面缺失,其余3种解离技术均可产生特征性碎片离子帮助判断二硫键。结论相较于传统的CID,EThcD在缩宫素多肽序列结构表征中具有明显优势,HCD和ETD具有技术互补性,单独使用CID尚不能完全表征其多肽结构。EThcD在含有二硫键的多肽类药物结构表征中具有较大的应用前景。OBJECTIVE To compare the performance of four kinds of dissociation methods in characterizing the peptide sequence of oxytocin by Orbitrap high-resolution mass spectrometry.METHODS The separation was performed on a fused silica capillary column(packed with C_(18)resin)with the mobile phase consisting of 0.1%formic acid aqueous solution(mobile phase A)and 0.1%formic acid aqueous solution–acetonitrile(20∶80)(mobile phase B)by gradient elution.The mass spectrometry was operated in the parallel reaction monitoring(PRM)under positive ion mode using electrospray ion source.Four kinds of dissocation methods,namely,collison induced dissociation(CID),higher-energy collision dissociation(HCD),electron transfer dissociation(ETD)and electron transfer/higher-energy collision dissociation(EThcD)were used to characterize the peptide sequence of oxytocin.RESULTS EThcD achieved the complete sequence coverage by producing the most abundant sequence informative ions related to peptide backbone.ETD achieved the moderate peptide sequencing,and ETD was slightly better than HCD,while CID performed the worst.Also,EThcD,ETD and HCD all produced characteristic ions relecting the disulfide bond in oxytocin,however,these ions were absent in CID mass spectra.CONCLUSION EThcD outperforms all other three dissociation methods in oxytocin sequence coverage.HCD and ETD can be complementary dissociation methods for peptide characterization.Using CID alone seems to be not adequade for oxytocin peptide sequencing.EThcD is a promising technique in peptide sequence for cyclopeptide with disulfide bond.

关 键 词：静电场轨道阱质谱 缩宫素 碰撞诱导解离 高能C阱碰撞诱导解离 电子转移解离 电子转移/高能碰撞诱导解离 

分 类 号：R917[医药卫生—药物分析学]