液质联用鉴定重组CHO细胞株目的蛋白的表达研究  

Study of liquid chromatography-mass spectrometry identification of target protein expression in recombinant CHO cell lines

作　　者：康赵飞 王可心 韩春乐 庞琳 饶春明 KANG Zhao-fei;WANG Ke-xin;HAN Chun-le;PANG Lin;RAO Chun-ming(JOINN Pharmaceutical Quality Research and Testing(Beijing)Co.,Ltd.,Beijing 102600,China)

机构地区：[1]北京昭衍药物检定研究有限公司,北京102600

出　　处：《药物分析杂志》2025年第1期99-103,共5页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:采用高分辨四极杆-飞行时间液质联用(LC-Q TOF MS)技术,探索重组中国仓鼠卵巢(CHO)细胞株目的蛋白的表达鉴定方法。方法:选择1株表达抗体的重组CHO细胞株,将该细胞悬液12500 r·min^(-1)离心10 min,取上清液,使用碳酸氢铵溶液(50 mmol·L^(-1))置换液体,加入胰凝乳蛋白酶(chymotrypsin)进行酶切,采用Advance Bio Peptide(100 mm×2.1 mm,2.7μm)色谱柱,通过Sciex TRIPLE TOF 5600+与Agilent Q TOF G65452台液质联用仪进行对比分析。根据保密要求和氨基酸覆盖率选择合适的特征肽段,并将该抗体目的蛋白重链与轻链的特征肽氨基酸序列分别输入SCIEX BioPharmaView Version 3.0与Agilent MassHunter BioConfirm 12.0软件中,建立数据库,对样品中实际检测得到的肽段进行搜库检索,验证并鉴定出目的蛋白特征肽。结果:样品通过Sciex TRIPLE TOF 5600+仪器分析得到8条特征肽段,氨基酸覆盖率为100%;样品通过Agilent Q TOF G6545仪器分析得到12条特征肽段,氨基酸覆盖率为100%。结论:本研究建立的方法操作简单,覆盖率高,稳定性好,可用于该抗体重组CHO细胞株目的蛋白的表达鉴定,也可为同类细胞株其他蛋白的表达鉴定提供参考。Objective:To optimize an LC-Q TOF MS method for the determination of target proteins expressed in recombinant cells from a cell bank.Methods:The recombinant Chinese hamster ovary(CHO)cell line expressing antibodies was selected.The supernatant of recombinant CHO cell suspensions was retained after 12500 r·min^(-1)centrifuging for 10 min and treated with 50 mmol·L^(-1)NH_(4)HCO_(3) solution.Chymotrypsin was added for enzymatic digestion,and the comparative analyses were carried out using two LC-Q TOF MS with Advance Bio Peptide(100 mm×2.1 mm,2.7μm)column,the Sciex TRIPLE TOF 5600+and the Agilent Q TOF G6545,respectively.To establish the database,the amino acid sequences of the characteristic peptides of the protein heavy and light chains were entered into SCIEX BioPharmaView Version 3.0 and Agilent MassHunter BioConfirm 12.0 software,respectively.The peptides detected in the samples were searched to identify the target peptides.Results:The samples were analysed by a Sciex TRIPLE TOF 5600+instrument to obtain 8 characteristic peptides with 100%amino acid sequence coverage and the samples were analysed by an Agilent Q TOF G6545 instrument to obtain 12 characteristic peptides with 100%amino acid sequence coverage.Conclusion:The method established in this study is simple to operate,has high coverage and good stability,can be used to identify the expression of target proteins in recombinant CHO cell lines.It also provides a reference for the expression identification of target proteins in similar cell lines.

关 键 词：重组蛋白 液质联用 氨基酸序列 特征肽鉴定 胰凝乳蛋白酶 胰蛋白酶 

分 类 号：R917[医药卫生—药物分析学]