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

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

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400~4000 m/z;扫描频率:2 Hz,锥孔电压:80 V;毛细管电压:3.0 kV;离子源温度:120℃。结果AAV衣壳蛋白VP1实测相对分子质量为81255.9,与理论值偏差为8.1 ppm;VP2蛋白实测相对分子质量为66062.9,与理论值偏差为3.8 ppm;VP3蛋白实测相对分子质量为59488.6,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。

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 400-4000 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 molecular mass of the AAV capsid proteins VP1,VP2,and VP3 was 81255.9,66062.9,and 59488.6,respectively.The deviations from the theoretical values were 8.1 ppm for VP1,3.8 ppm for VP2,and 36 ppm for VP3.Mass peptide profile analysis 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 RPLCMS 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.