反相液相色谱-质谱联用技术对重组腺相关病毒6型衣壳蛋白的表征分析

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中文摘要：目的应用反相液相色谱-质谱(reversed phase liquid chromatography-mass spectrometry,RPLC-MS)联用技术对重组腺相关病毒6型(recombinant adeno-associated virus 6,rAAV6)载体的衣壳蛋白进行表征分析,包括一级结构和翻译后修饰(post-translational modification,PTM).方法流动相 A为0.1％二氟乙酸(difluoroacetic acid,DFA)的水溶液;流动相B为0.1％DFA的乙腈溶液;柱温:80 ℃;梯度洗脱10 min(0→10 min,流动相B 15％→45％).ESI-Q-TOF质谱检测设为阳离子模式:质荷比扫描范围400～4000m/z;扫描频率:2 Hz,锥孔电压:80 V;毛细管电压:3.0kV;离子源温度:120 ℃.结果 AAV衣壳蛋白VP1实测相对分子质量为81 255.9,与理论值偏差为8.1 ppm;VP2蛋白实测相对分子质量为66 062.9,与理论值偏差为3.8 ppm;VP3蛋白实测相对分子质量为59 488.6,与理论值偏差为36 ppm.rAAV6酶切后质量肽图分析,样品序列覆盖率为89％,检测到的PTM主要包括脱酰胺、N-末端乙酰化、泛素化、磷酸化等,并未发现糖基化修饰位点.通过串联质谱分析确证了rAAV6衣壳蛋白N-末端和C-末端的序列以及N-末端的PTM.结论应用RPLC-MS联用技术分析了rAAV6衣壳蛋白的完整相对分子质量,并在肽段水平运用串联质谱深度分析了 rAAV6衣壳蛋白的PTM,对AAV基因治疗制品的质量控制及生产工艺的提高具有一定意义.

外文标题：Characterization analysis of capsid protein of recombinant adeno-associated virus 6 using reversed phase liquid chromatography-mass spectrometry

外文摘要：Objective To characterize the capsid proteins of recombinant adeno-associated virus 6(rAAV6)vectors by reversed phase liquid chromatography-mass spectrometry(RPLC-MS),including primary structure and post-translational modification(PTM).Methods The mobile phase A consisted of 0.1％aqueous solution of difluoroacetic acid(DFA),while the mobile phase B was 0.1％DFA acetonitrile solution.The column temperature was maintained at 80 ℃,and the gradient elution lasted for 10 min(0→10 min,mobile phase B 15％→45％).The ESI-Q-TOF mass spectrometry detection operated in positive ion mode with the scanning range of 400-4 000 m/z,the scanning frequency of 2 Hz,the cone voltage at 80 V,the capillary voltage at 3.0 kV,and the ion source temperature at 120 ℃.Results The measured relative molecu-lar mass of the AAV capsid proteins VP1,VP2,and VP3 was 81 255.9,66 062.9,and 59 488.6,respectively.The devia-tions from the theoretical values were 8.1 ppm for VP1,3.8 ppm for VP2,and 36 ppm for VP3.Mass peptide profile analy-sis of the enzymatically digested rAAV6 sample indicated a sequence coverage of about 89％ with detected PTMs mainly including deamidation,N-terminal acetylation,ubiquitination,and phosphorylation;no glycosylation modification sites were found.Tandem mass spectrometry confirmed the N-terminal and C-terminal sequences of the rAAV6 capsid protein as well as the N-terminal PTM.Conclusion The complete relative molecular mass of rAAV6 capsid protein was analyzed by RPLC-MS technique,and the PTM of rAAV6 capsid protein was analyzed by tandem mass spectrometry at the peptide level,which has a certain significance for the quality control of AAV gene therapy products and the improvement of production process.

外文关键词：

Adeno-associated virus(AAV)Capsid proteinReversed phase liquid chromatography(RPLC)Tandem mass spectrometry(MS/MS)Relative molecular massPeptide mapping

作者：

郭莹、李响、张拓、史新昌、何娟、聂爱英、裴德宁、周勇、秦玺、梁成罡

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作者单位：

中国食品药品检定研究院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生物制品质量研究与评价重点实验室,北京 100050

沃特世科技(上海)有限公司,上海 200126

关键词：

腺相关病毒衣壳蛋白反相液相色谱串联质谱相对分子质量肽图

基金：

国家重点研发计划中国医学科学院中央级公益性科研院所基本科研业务费专项资金国家质量基础设施体系资助

项目编号：

2023YFC34033052023-PT350-012021YFF0600804

出版年：

2024

中国生物制品学杂志

中华预防医学会,长春生物制品研究所有限责任公司

中国生物制品学杂志

CSTPCD

影响因子：0.417

ISSN：1004-5503

年,卷(期)：2024.37(7)