人乳头瘤病毒18型L1抗原的肽图特征峰分析

Analysis of characteristic peaks of HPLC peptide profiles of human papillomavirus type 18 L1 antigen

目的:比较分析不同氨基酸序列人乳头瘤病毒(HPV)18型L1抗原的肽图,收集差异特征峰肽段,利用质谱方法对其进行鉴别。方法:将HPV18型L1抗原用胰蛋白酶水解,酶解所得的肽段通过RP-HPLC法分离肽段,得到HPV18型L1抗原肽图;用Megalign软件比较不同来源的L1抗原的氨基酸序列差异,分析不同HPV18型L1抗原的肽图特征峰,收集差异特征峰;HPLC色谱条件:采用C18(4.6 mm×250 mm,5μm)色谱柱,以0.1%三氟乙酸-水为流动相A,0.1%三氟乙酸-乙腈为流动相B,梯度洗脱(0~10 min,100%A;10~80 min,100%A→30%A;80~91 min,30%A→100%A),流速1.0mL·min-1,柱温30℃,检测波长214 nm,进样量100μL。再运用UPLC-MS/MS技术,采用电喷雾离子源(ESI),正离子全扫描检测模式,喷雾电压2 kV,毛细管温度300℃,对差异特征峰进行鉴别。结果:5个公司生产的L1抗原平行6次重复检测,各共有峰保留时间的RSD最大值范围为0.24%~0.49%,表明肽图分析方法重复性良好。对5个公司的3批次抗原进行检测,主要峰保留时间的最大RSD范围为0.20%~0.74%,表明批间一致性良好。比较发现5个不同公司生产的HPV疫苗18型L1抗原的液相肽图有6个差异特征峰(1~6号),分别对其进行质谱鉴定,结合氨基酸序列的比对分析,发现5个特征峰由氨基酸差异引起:487~496位氨基酸的缺失将导致1号特征峰的缺失;88位氨基酸N和T的相互转变导致2、3号特征峰相互转变;494位氨基酸S和A的相互转变导致1、4号特征峰相互转变;5号特征峰为4号特征峰胰蛋白酶酶切不完全出现的;6号峰为非特异峰。表明不同公司生产的HPV18型L1抗原具有各自的特征峰。结论:本研究建立的HPLC-MS/MS肽图特征峰分析方法,可应用于不同企业生产的18型的HPV L1抗原的鉴别和质量控制,进一步表明了肽图在疫苗原液质量控制中的重要意义。

Objective:To collect and identify the characteristic peaks by mass spectrometry,compare and analyze the peptide mappings of human papillomavirus(HPV)18 L1 antigen of different amino acid sequences. Methods:HPV18 L1 antigen was digested by trypsin,and the enzymatic digested peptide segments were analyzed by RPHPLC to obtain the peptide map of HPV18 L1 antigen. The amino acid sequences differences of L1 antigen among different companies were compared with Megalign software,and the characteristic peaks of different HPV18 L1 antigens were analyzed to collect the characteristic peaks. The chromatographic conditions of HPLC were as followed:C18 column(4.6 mm×250 mm,5 μm)was adopted,using 0.1% trifluoroacetic acid-water as mobile phase A,0.1% trifluoroacetic acid-acetonitrile as mobile phase B,with gradient elution(0-10 min,100%A;10-80 min,100%A → 30%A;80-91 min,30%A → 100%A)at flow rate of 1.0 mL·min-1,the column temperature was 30 ℃,the detection wavelength was 214 nm,and the injection volume was 100 μL. The characteristic peaks of HPV18 L1 antigen were collected and identified by UPLC-MS/MS. Then UPLC-MS/MS technology was used to identify the differential characteristic peaks using electrospray ion source(ESI),positive ion scanning detection mode,spray voltage 2 kV,capillary temperature 300 ℃. The UPLC-MS/MS technology was used to identify the differential characteristic peaks using electrospray ion source(ESI),positive ion full scan detection mode,spray voltage 2 kV and capillary temperature 300 ℃. Results:L1 antigens produced by five companies were detected six times in parallel. The maximum RSD of retention time of each common peak ranged from 0.24% to 0.49%,indicating that the method of peptide map analysis was in good repeatability. Three batches of antigens from five companies were tested,and the maximum RSD of main peaks retention time was 0.20% to 0.74%,indicating good consistency among batches. Comparing the HPLC peptide mapping results of L1 antigen of HPV vaccine produced by five different companies,six differential characteristic peaks(No. 1-6)were collected and identified by mass spectrometry. Combined with the comparative analysis of amino acid sequences,5 characteristic peaks were found to be caused by amino acid sequence differences. The loss of 487-496 amino acids would lead to the loss of characteristic peak No.1. The mutual transformation of amino acid N and T at position 488 resulted in the transformation of characteristic peaks No. 2 and No. 3. The transformation of amino acids S and A at position 494 resulted in the transformation of characteristic peaks No. 1 and No. 4. The peak No. 5 was the characteristic peak of incomplete enzymatic digestion of characteristic peak No. 4. Peak No. 6 was non-specific peak appearing in A-D four companies’ products. These results indicated that HPV18 L1 antigen produced by different companies had their own characteristic peaks of HPLC peptide profiles. Conclusion:The characteristic peaks of HPLC-MS/MS peptide profiles established in this study can be applied to the identification and quality control of HPV type 18 L1 antigen,further demonstrating the importance of the peptide mapping method in the quality control of vaccine stock solution.