利用质谱技术对肝癌组织中自然呈递的MAGE表位进行鉴定

Identification of naturally presented MAGE epitopes from tissues of hepatocellular carcinoma by mass spectrometry

目的:利用质谱检测技术和差异比较方法,对肝癌组织中自然呈递的黑色素瘤抗原基因(melanomaantigengene,MAGE)表位进行鉴定分析.方法:肝癌细胞和癌旁无瘤肝细胞取自同1例肝癌患者,弱酸洗涤法分离肝癌细胞和肝细胞表面所有肽段;利用表位预测法挑选出HLA—A2限制的MAGE-1,MAGE-3理论侯选肽作为质谱筛选目标;利用弱酸洗涤法从肝癌细胞和肝细胞表面分离肽段,这些肝癌细胞和肝细胞都来自同1例肝癌患者.然后用HPLC分离纯化这些肽段,并将二种细胞的各峰段(fractions)进行差异比较,挑选出肿瘤特异性的峰段进行质谱分析.结果:经表位预测,共选出80条目标肽.从肝癌细胞样品中检测出2条自然呈递的MAGE抗原肽:FLWGPRALV(MAGE-3271-279)和FPSLREAAL(MAGE-1294-302),他们分别来自HCC的峰45.246和峰34.801,m/z分别为1058.49和1003.62.结论:这是首次从肿瘤组织中分离、鉴定出自然呈递的MAGE抗原表位.本实验证实质谱检测法可以对组织中自然呈递的肿瘤相关抗原表位进行快速准确检测,而检测的准确性和高效性对于表位的鉴定和肿瘤疫苗的设计部是非常重要的.

AIM: To detect the naturally processed antigenic peptides of melanoma antigen gene (MAGE) in tissues of hepato-cellular carcinoma (HCC) by the technology of mass spec-trometry (MS) and the strategy of differential analysis. METHODS: Epitope prediction was performed to select HLA-A2-restricted protein sequence of MAGE-1 and MAGE-3 (theoretic peptides) to serve as the targets for MS screening. Peptides were isolated by mild acid elu-tion from the surfaces of HCC cells and non-neoplastic liver cells, which were obtained from the same patient with HCC. Then the mixtures of peptides were fractionated by HPLC and a differential comparison was made between the two kinds of cells. Finally, the fractions contained tumor-specific peptides were analyzed by MS. RESULTS: Eighty target peptides were selected after epitope prediction. Two naturally processed peptides of MAGE antigen were detected from HCC samples: FLWGPRALV (MAGE-3271-279) and FPSLREAAL(MAGE-1294-302). They were from peak 45.246 and 34.801, and the m/z was 1 058.49 and 1 003.62 respectively. CONCLUSION: It is the first evidence that naturally presented peptides of MAGE antigen can be isolated and identified from the tumor tissues. MS-based approach can determine naturally processed peptides of tumor antigen rapidly and precisely, which is important in epitope identification and vaccine designation.