Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high performance liquid chromatography electrospray ionization mass spectrometry
Structural characterization of site-specific N-glycosylation of recombinant human factor VIII by reversed-phase high performance liquid chromatography electrospray ionization mass spectrometry
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DOI：
10.1021/ac970372z
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发表时间：
1997-10-01
影响因子：
7.4
通讯作者：
Burlingarne, AL
中科院分区：
文献类型：
--
作者：
Medzihradszky, KF;Besman, MJ;Burlingarne, AL
本研究探讨了在中国仓鼠卵巢细胞中表达的重组人凝血因子VIII中N-连接寡糖的位点占用率和位点特异性碳水化合物微异质性。分离与人凝血酶孵育后形成的四种因子VIIIa多肽，并分别用胰蛋白酶进行蛋白水解。通过反相高效液相色谱/电喷雾电离质谱法分析这些胰蛋白酶促消化物。利用诊断性碳水化合物离子的选择离子监测来鉴定含糖肽的色谱峰。在50和73 kDa多肽的糖基化位点处检测到低聚甘露糖和复合碳水化合物，而在B结构域上鉴定的所有低聚糖均为复合型结构。只有43 kDa的多肽被发现非糖基化。这些研究确立了双触角核心岩藻糖基化碳水化合物作为主要取代基，这与对整个N-连接碳水化合物库的分析结论一致（Kumar，H.下午;海牙，C.; Haley，T.;斯塔尔角M.; Besman，M. J.道：Lundblad，R.;贝克，D。Biotechnol. 1996，24，207-216）。此外，该质谱研究显示存在以前未报道的该糖蛋白的复杂非岩藻糖基化低聚糖。
The present study addresses the site occupancy and the site-specific carbohydrate microheterogeneity of N-linked oligosaccharides in recombinant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thrombin, were isolated and separately subject to proteolysis with trypsin. These tryptic digests were analyzed by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Selected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose and complex carbohydrates were detected at the glycosylation sites of the 50 and 73 kDa polypeptides, while all the oligosaccharides identified on the B-domain were complex-type structures. Only the 43 kDa polypeptide was found nonglycosylated. These studies established a biantennary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydrate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 207-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.