利用高分辨质谱技术综合解析胰岛素类制品复杂二硫键结构  

Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum

作　　者：胡馨月 丁晓丽[1] 孙悦[1] 张慧[1] 李晶[1] 梁成罡[1] HU Xin-yue;DING Xiao-li;SUN Yue;ZHANG Hui;LI Jing;LINAG Cheng-gang(Division of Hormone,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院激素室,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局化学药品质量研究与评价重点实验室,北京102629

出　　处：《药学学报》2024年第1期188-197,共10页Acta Pharmaceutica Sinica

基　　金：中国食品药品检定研究院中青年发展研究基金课题(2022A3);国家重点研发计划课题(2021YFF0600804)。

摘　　要：二硫键的正确配对维持了多肽和蛋白类药物的正确折叠方式和高级结构的形成,对产品的质量控制至关重要。为了确保二硫键正确配对,二硫键分析是多肽和蛋白质药物表征中必不可少的重要部分。质谱分析技术可用于二硫键分析,然而,胰岛素及其类似物中存在两对没有酶切位点的二硫键,常规的碰撞诱导解离(CID)和高能诱导裂解(HCD)无法实现该复杂二硫键的准确定位。本研究通过3种方法综合定位该复杂二硫键,包括酶切加关键肽段源内碎裂(ISD)法、酶切加部分还原烷基化法、完整蛋白源内碎裂和电子转移解离(ETD)裂解法,并且考察了门冬胰岛素、赖脯胰岛素和甘精胰岛素的适用性,为胰岛素及其类似物二硫键连接方式的质量控制提供了新途径,也为含该类复杂二硫键多肽或蛋白类生物制品的二硫键定位提供借鉴。The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs,which is crucial for the quality control of products.In order to ensure that the disulfide bonds are correctly paired,disulfide bond analysis is an essential part of peptides and protein drug characterization.Mass spectrometry can be used to analyze disulfide bonds.However,insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site.Conventional collision-induced dissociation(CID)and high-energy induced cleavage(HCD)cannot accurately locate the complex disulfide bond.In our study,three methods were used to localize the complex disulfide,including enzyme digestion combined with key peptide fragment in source decay(ISD)fragmentation method,enzyme digestion combined with partial reduction alkylation method,intact protein source ISD and electron transfer dissociation(ETD)cleavage method,The applicability of insulin aspart,insulin lispro and insulin glargine were also investigated.This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues,and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

关 键 词：胰岛素及其类似物 二硫键定位 部分还原烷基化 源内碎裂 电子转移裂解 

分 类 号：R917[医药卫生—药物分析学]