酶切质谱法和氰基化裂解法联合解析新药重组人门冬胰岛素二硫键  

Combination of enzymatic digestion- mass spectrometry and cyanylation cleavage to analyze disulfide bonds of new drug recombinant human insulin aspart

作　　者：黄亚娟[1,2] 傅海媛[2] 侯利平[1,2] 孙云波[2] 张拓[2] 王东茂[2] 郑俊杰[2] 魏开华[2] 

机构地区：[1]中南大学药学院,长沙410013 [2]军事医学科学院放射与辐射医学研究所,北京蛋白质组研究中心,蛋白质组学国家重点实验室,国家蛋白质科学中心(北京),北京102206

出　　处：《药物分析杂志》2013年第10期1649-1655,共7页Chinese Journal of Pharmaceutical Analysis

基　　金：863项目(2012AA020205);科技部重大仪器专项(2012YQ180117)

摘　　要：目的:建立一种简化、快速、准确的分析含有相邻Cys的双链型二硫键的方法,并确定重组人门冬胰岛素的二硫键。方法:(1)ESI-Q-TOF质谱准确测定样品还原烷基化及烷基化前后的相对分子质量,计算二硫键数和自由巯基数;(2)采集Glu-C酶切肽质量指纹谱,MS-Bridge检索并确定1对链间二硫键;(3)简化氰基化裂解法步骤,LC-MS/MS测定关键肽段序列,自编软件结合人工解析,确定第2、3对二硫键。结果:(1)相对分子质量测定出重组人门冬胰岛素含3对二硫键;(2)酶切肽谱的序列覆盖率为100%,关键肽段m/z 1377.5568、m/z 2493.1045,测量偏差小于0.05,对应二硫键A20-B19。(3)Glu-C酶切液经氰基衍生后以ZipTip除盐代替HPLC制备,氨水裂解后用MALDI-TOF测肽谱,获得关键肽段m/z1530.7163,对应二硫键A6-A11,其串联质谱共匹配14个关键序列离子,碎片离子强度高,质量偏差小于0.01,证实该二硫键连接正确。结论:新药重组人门冬胰岛素主要的二硫键连接方式为A6-A11、A7-B7、A20-B19,与已知胰岛素一致。本方法降低了双链连接型胰岛素二硫键分析复杂度,简化了步骤,具有良好的实用性。Objective： To establish a simplified,rapid and reliable method for analysis of double-chain disulfide bond containing adjacent Cys and determine the disulfide bond of recombinant human insulin aspart. Methods： （1） The relative molecular mass before and after reduced alkylation and alkylation was measured accurately and the number of disulfide bond and free sulfhydryl was calculated.（2） Glu-C digested peptide mass fingerprinting （PMF） was collected and the search with MS-Bridge was performed to determine a pair of inter-chain disulfide bond.（3） Steps of cyanylation cleavage were simplified,the sequence of key peptides was measured by LC-MS/MS,and self-developed software was used in combination with manual interpretation to determine the second and third disulfide bonds. Results： （1） There were three disulfide bonds in recombinant human insulin aspart through measurement of relative molecular mass.（2） The coverage of sequence in PMF was 100%.Key peptides m/z 1377.5568 and m/z 2493.1045,which corresponded to disulfide bond A20-B19,were detected;the measurement errors were less than 0.05.（3） After Glu-C digestion went through cyno-derivation,HPLC preparation was replaced by desalination by ZipTip.Ammonia cleavage PMF was measured by MALDI-TOF,and the key peptide m/z 1530.7163 which corresponded to disulfide bond A6-A11 was detected and then submitted to MS/MS analysis,and 14 key sequence ions with high ion intensity were matched,mass errors were less than 0.01,these results verified the disulfide bond of A6-A11. Conclusion： The major disulfide bonds of the new drug recombinant human insulin aspart are A6-A11,A7-B7,and A20-B19,which are consistent with those of the natural insulin.This method reduces analytical complexity of double-chain insulin,simplifies the procedure and shows good practicability.

关 键 词：重组人门冬胰岛素 二硫键定位解析 半胱氨酸 肽质量指纹图谱 氰基化裂解 质谱分析 

分 类 号：R917[医药卫生—药物分析学]