HUMAN ALPHA-FETOPROTEIN PRODUCED FROM HEP G2 CELL-LINE - STRUCTURE AND HETEROGENEITY OF THE OLIGOSACCHARIDE MOIETY
HUMAN ALPHA-FETOPROTEIN PRODUCED FROM HEP G2 CELL-LINE - STRUCTURE AND HETEROGENEITY OF THE OLIGOSACCHARIDE MOIETY
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DOI：
10.1002/jms.1190300415
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发表时间：
1995-04-01
影响因子：
2.3
通讯作者：
MALORNI, A
中科院分区：
文献类型：
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作者：
FERRANTI, P;PUCCI, P;MALORNI, A
用高分辨色谱技术和质谱仪相结合的方法研究了人甲胎蛋白的糖基组成。甲胎蛋白是肝癌细胞系Hep G2产生的一种RMM 67000糖蛋白。快速原子轰击质谱仪(FABMS)和反相高效液相色谱(RP-HPL C)分析表明，甲胎蛋白经胰酶和N-聚糖酶F水解后，在天冬酰胺232水平上含有单一的糖基化位点。此外，对完整蛋白质分子质量的电喷雾质谱测量表明，存在两种主要的糖形式。通过高效阴离子交换层析和脉冲安培检测，结合FABMS对释放的寡糖进行碳水化合物作图，完整地定义了寡糖部分的结构异质性。结果表明：(I)细胞培养产生的糖基化是典型的人肝癌甲胎蛋白的双天线复合型糖基化；(Ii)存在两种主要结构，比例约为2：1，在分子的非还原部分，N-乙酰氨基葡萄糖中存在岩藻糖残基。
The carbohydrate moiety of human alpha-fetoprotein, an RMM 67 000 glycoprotein produced in a hepatoma cell line (Hep G2), was investigated by the combined use of high-resolution chromatographic techniques and mass spectrometry. Fast atom bombardment mass spectrometric (FABMS) and reversed-phase high-performance liquid chromatographic analysis of alpha-fetoprotein obtained from a large-scale cell culture following tryptic and peptide N-glycanase F hydrolysis demonstrated that the protein contains a single glycosylation site at level of asparagine 232. Further, electrospray mass spectrometric measurement of the intact protein molecular mass showed that two main glycoforms are present The complete definition of the structural heterogeneity of the oligosaccharide moiety was achieved by high-performance anion-exchange chromatography with pulsed amperometric detection together with carbohydrate mapping by FABMS of the released oligosaccharides demonstrating (i) that the glycosylation produced by cell culture is of the biantennary complex type typical of human hepatoma alpha-fetoprotein and (ii) the presence of two main structures in a ratio of about 2:1 differing in the presence of a fucose residue in the N-acetylglucosamine in the non reducing portion of the molecule.