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12 500 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, 2.7 μm）色谱柱,通过Sciex TRIPLE TOF 5600+与Agilent Q TOF G6545 2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 12 500 r · min-1centrifuging for 10 min and treated with 50 mmol · L-1 NH4HCO3 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, 2.7 μm) 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.
[1] BODDAPATI S, GILMORE J, BOONE K, et al.Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines[J]. PLoS One, 2020, 15(10): e0241250
[2] FISCHER S, HANDRICK R, OTTE K.The art of CHO cell engineering: a comprehensive retrospect and future perspectives[J]. Biotechnol Adv, 2015, 33(8): 1878
[3] KUO CC, CHIANG AW, SHAMIE I, et al.The emerging role of systems biology for engineering protein production in CHO cells[J]. Curr Opin Biotechnol, 2018, 51: 64
[4] TIHANYI B, NYITRAY L.Recent advances in CHO cell line development for recombinant protein production[J]. Drug Discov Today Technol, 2021, 38: 25
[5] 吴海涛,胡云龙,陈松,等. 重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素[J]. 中国生物工程杂志,2004,24(8): 1
WU HT, HU YL, CHEN S, et al.The factors influencing the high-level expression of recombinant protein in CHO cells[J]. China Biotechnol, 2004, 24(8): 1
[6] 高凯,陶磊,王军志. 重组抗体药物的质量控制[J]. 中国新药杂志,2011,20(19): 1848
GAO K, TAO L, WANG ZJ.Quality control for recombinant therapeutic antibodies[J]. Chin J New Drugs, 2011, 20(19): 1848
[7] 高凯,任跃明,王兰,等. 关于我国药典重组DNA技术产品总论的思考[J]. 中国生物工程杂志,2014,34(5): 107
GAO K, REN YM, WANG L, et al.Points to consider for the general monograph of products prepared by recombinant DNA technology in Chinese Pharmacopeia[J]. China Biotechnol, 2014, 34(5): 107
[8] 中华人民共和国药典2020年版. 三部[S]. 2020: 11
ChP 2020. Vol Ⅲ[S]. 2020: 11
[9] BECCARIA M, CABOOTER D.Current developments in LC-MS for pharmaceutical analysis[J]. Analyst, 2020, 145(4): 1129
[10] FANG ZZ, GONZALEZ FJ.LC-MS-based metabolomics: an update[J]. Arch Toxicol, 2014, 88: 1491
[11] BALDWIN MA.Protein identification by mass spectrometry: issues to be considered[J]. Mol Cell Proteomics, 2004, 3(1): 1
[12] GATLIN CL, ENG JK, CROSS ST, el al. Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry[J]. Anal Chem, 2000, 72: 757
[13] STEVENS SM JR, KEM WR, PROKAI L.Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques[J]. Rapid Commun Mass Spectrom, 2002, 16(22): 2094
[14] 陶磊,于雷,丁有学,等. 液质联用分析重组人尿激酶原的糖基修饰异质性[J]. 药学学报,2020,55(11): 2713
TAO L, YU L, DING YX, et al.Analysis of the glycosylation heterogeneity of recombinant human pro-urokinase using UPLC-MS[J]. Acta Pharm Sin, 2020, 55(11): 2713
[15] ZHANG X, JIN X, LIU L, et al.Optimized reversed-phase liquid chromatography/mass spectrometry methods for intact protein analysis and peptide mapping of adeno-associated virus proteins[J]. Hum Gene Ther, 2021, 32(23-24): 1501
[16] YU YQ, GILAR M, LEE PJ, et al.Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins[J]. Anal Chem, 2003, 75(21): 6023
