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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。
AIM To identify one HLA-A2-restricted MAGE-A3 epitope FLWGPRALV (MAGE-A3_ (271-279)) naturally presented on the surface of human hepatoma cell line HLE cells. Methods The synthetic peptide FLWGPRALV was identified by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (HPLC-MS / MS), and the HPLC peak time and mass spectrometry signal characteristics of the peptide were identified. The detection limit of the two peptides . The antigen peptides naturally expressed by the major histocompatibility complex antigen (MHC) molecules were eluted from the surface of HLE cells by weak acid washing method, purified by HPLC, and fractionated according to the peak time of artificial synthetic peptides 2 min fractions, low temperature freeze-dried by liquid chromatography-mass spectrometry to identify whether there MAGE-A3_ (271-279) antigen polypeptide. Results The purified peptide sample was detected by liquid chromatography-mass spectrometry (MS / MS), and a (m / z) _2 529.9 double-charge mass spectrometry signal was obtained. The collision-induced dissociation spectrum was further analyzed to prove that its sequence To C-terminus) was FLWGPRALV with a relative molecular mass of 1058.4 Da, which was in agreement with the MAGE-A3_ (271-279) antigenic peptide, indicating the presence of a low abundance of the antigenic polypeptide of interest in the sample. Conclusion The MAGE-A3 epitope peptide, which can be naturally presented by HLA-A2 molecules, can be directly isolated from the surface of human hepatoma cell lines, suggesting that the MAGE-A3 271-279 polypeptide has potential application in the immunotherapy of hepatocellular carcinoma.