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

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/z 1530.7163,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.