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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 simple, rapid and accurate method for the analysis of double-chain disulfide bonds containing adjacent Cys and to determine the disulfide bond of recombinant human insulin aspart. Methods: (1) ESI-Q-TOF mass spectrometry was used to accurately determine the relative molecular mass of the samples before and after reductive alkylation and alkylation. The number of disulfide bonds and free thiols were calculated. (2) The quality fingerprints of Glu- (3) To simplify the steps of cyanation, LC-MS / MS determination of the key peptide sequence, self-compiled software combined with manual analysis to determine the first 2,3 Disulfide bond. Results: (1) The relative molecular mass was determined to contain 3 pairs of disulfide bonds in the recombinant human insulin aspart. (2) The sequence coverage of the digested peptide was 100%. The key peptides m / z 1377.5568, m / z 2493.1045, The measurement deviation is less than 0.05, corresponding to disulfide bonds A20-B19. (3) Glu-C enzyme digestion by cyano derived ZipTip desalination instead of HPLC preparation, ammonia cleavage by MALDI-TOF measured peptide spectrum, the key peptide m / z1530.7163, corresponding to the disulfide bond A6-A11 The tandem mass spectrometry matched a total of 14 key sequence ions. The fragment ion intensity was high and the mass deviation was less than 0.01, confirming that the disulfide bond was correct. CONCLUSION: The main disulfide bonding mode of insulin as a new recombinant human aspart insulin is A6-A11, A7-B7 and A20-B19, which are consistent with known insulin. The method reduces the complexity of double-linked insulin disulfide analysis, simplifies the procedure and has good practicability.