利用质谱技术鉴定人肝癌细胞表面自然呈递的MAGE—A3表位

Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry

目的鉴定人肝癌细胞系 HLE 细胞表面自然呈递的1条 HLA-A2限制性 MAGE-A3抗原表位 FLWGPRALV(MAGE-A3_(271-279))。方法用高效液相色谱仪(HPLC)及液-质联用仪对人工合成多肽 FLWGPRALV 进行鉴定,明确该多肽的 HPLC 出峰时间和质谱信号特征,并确定这两种仪器对该多肽的检测下限。以弱酸洗涤法从 HLE 细胞表面洗脱出由主要组织相容性抗原复合物(MHC)分子自然呈递的抗原多肽,用 HPLC 纯化分馏,根据人工合成多肽的出峰时间,收集该出峰时间前后2 min 的馏分,低温冻干后以液.质联用仪进行鉴定,明确其中是否存在 MAGE-A3_(271-279)抗原多肽。结果经纯化的多肽洗涤样品经液.质联用仪检测,可获得一个(m/z)_2为529.9的双电荷质谱信号,对其碰撞诱导解离谱图进一步分析,证明其序列(自 N 端至 C 端)为 FLWGPRALV,相对分子质量为1058.4 Da,与 MAGE-A3_(271-279)抗原多肽相符,说明样品中存在低丰度的目的抗原多肽。结论可以从人肝癌细胞系表面直接分离、鉴定出可被 HLA-A2分子自然呈递的 MAGE-A3表位多肽,提示 MAGE-A3_(271-279)抗原多肽在肝癌免疫治疗中具有潜在应用价值。

Objective To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV （MAGE-A3271-279）, on the surface of a human hepatocellular carcinoma （HCC） cell line HLE. Methods Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 × 10^9 HLE cells were collected, peptides naturally presented by major histocompatibility complex （MHC） molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 rain after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3271-279 peptide. Results The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of （m/z）2 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE- A3271-279 peptide,its amino sequence was FLWGPRALV and its molecular weight was 1058. 4 Da. Conclusions MAGE-A3271-279 epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3271-279 peptide may have potential immunotherapeutic value in HCC patients.