基于电喷雾电离质谱的人肝癌细胞HepG2与正常肝细胞L02的N-糖链的定性定量比较

以培养的原发性肝癌细胞HepG2和正常肝细胞L02为研究对象,采用细胞裂解液提取总蛋白,用PNGase F酶解释放N-糖链,以微晶纤维素柱结合石墨碳柱纯化分离N-糖链,通过电喷雾电离质谱(ESI-MS)和串联质谱(MS/MS)对N-糖链进行序列鉴定,以β-环糊精为内标对2种细胞系的N-糖链进行了定量比较分析.结果表明,在肝癌细胞系HepG2和正常细胞系L02中共检测到26种N-糖链,与L02相比,HepG2的大多数高甘露糖型糖链、唾液酸化糖链和岩藻糖基化糖链的数量都明显升高,其中有15种糖链在数量上具有极显著性差异(p0.01),1种糖链具有显著性差异(p0.05).本研究为进一步探索肝癌中各类N-糖链的表达特点及发现早期肝癌糖链标志物提供了参考.

Qualitation and Quantitation for Comparative Analysis of N-glycans from Human Hepatocellular Carcinoma HepG2 and Normal Liver Cells L02 by Electrospray Ionization Mass Spectrometry

HepG2, a primary hepatocellular carcinoma cell line, and L02 derived from normal liver tissue, were chosen as model cell lines. In this work, the total proteins of HepG2 and L02 cells were extracted and enzymatically hydrolyzed by Peptide-N-glycosidase F(PNGase F). Then the released N-glycans were purified by microcrystalline cellulose and graphitized carbon columns. The structure of the purified N-glycans was identified by electrospray ionization mass spectrometry(ESI-MS)and MS/MS. To compare the difference of N-glycans between HepG2 and L02 cells, β-cyclodextrin was used as the internal standard to relative quantitative analysis of the N-glycans by MS. As results, 26 N-glycans are observed in both HepG2 and L02 cells. The amounts of high-mannose, sialylated, as well as fucosylated and sialylated N-glycans of HepG2 were higher than those of L02 cells. Among them, 15 kinds of N-glycans of HepG2 cells show very significant difference(p<0.01) and 1 kind of N-glycan show significant difference(p<0.05), compared to those of L02 cells. Our studies show potential in investigation of structure partterns of N-glycans expressed in hepatocellular carcinoma and in finding early biomarker in liver cancer diagnose.