固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

引用本文: 刘让东, 许歆瑶, 王薇薇, 王彦, 闫超. 固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用[J]. 色谱, 2019, 37(10): 1090-1097. doi: 10.3724/SP.J.1123.2019.03019 shu

Citation: LIU Rangdong, XU Xinyao, WANG Weiwei, WANG Yan, YAN Chao. Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins[J]. Chinese Journal of Chromatography, 2019, 37(10): 1090-1097. doi: 10.3724/SP.J.1123.2019.03019 shu

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

上海交通大学药学院, 上海 200240
基金项目:
国家自然科学基金项目（21874088，81874307）；上海市科委"科技创新行动计划"（16142200300，17142201000，18142200700）；上海市标准化推进专项（17DZ2201500）.

摘要: 通过聚合物原位聚合反应，制备了部分填充的毛细管整体柱。pH 3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上，构建了适用于固化pH梯度毛细管等电聚焦整体柱（M-IPG）的平台。在蛋白质药物测定过程中，用M-IPG柱和羟丙基纤维素（HPC）涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明，两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点（pI），M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致，表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。

关键词:

English

Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
Received Date: 16 March 2019
Fund Project:
National Natural Science Foundation of China (Nos. 21874088, 81874307), Shanghai Science and Technology Commission Scientific Research Project (Nos. 16142200300, 17142201000, 18142200700)

Abstract: A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface. For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit. Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (pI) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the pI values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary. It was found that these two cIEF columns can be effectively used to separate proteins and determine the pI values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured pI values were consistent with those estimated using the HPC capillary.

Key words:

1. [1] Schartmann B, Gattner H G, Danho W, et al. Hoppe-Seylers Z Physiol Chem, 1983, 364:179
2. [2] Westin O, Rasmussen L J H, Andersen O, et al. J Atrial Fibril, 2018, 10:1801
3. [3] Anderson C M, Sonis S T, Lee C M, et al. Int J Radiat Oncol Biol Phys, 2018, 100:427
4. [4] Chen W, Taché Y, Marvizón J C. Neuroscience, 2018, 381:149
5. [5] Fujinaga S, Nishino T, Umeda C, et al. Pediatr Nephrol, 2019, 34:353
6. [6] Cabral T, Lima L H, Mello L G M, et al. Ophthalmol Retina, 2018, 2:31
7. [7] Kundu S, Pandit S, Abbas S, et al. Chem Phys Lett, 2018, 693:176
8. [8] Dai J, Lamp J, Xia Q, et al. Anal Chem, 2018, 90:2246
9. [9] Wang L, Bo T, Zhang Z, et al. Anal Chem, 2018, 90:9495
10. [10] Srivorakun H., Fucharoen G, Sanchaisuriya K, et al. Int J Lab Hematol, 2017, 39:101
11. [11] Meng X G, Zhen L, Liu Y M, et al. Journal of Instrumental Analysis, 2017(3):343 孟晓光, 甄里, 刘悦玫, 等. 分析测试学报, 2017(3):343
12. [12] Dauriac V, Descroix S, Chen Y, et al. Electrophoresis, 2008, 29:2945
13. [13] Herr A E, Molho J I, Drouvalakis K A, et al. Anal Chem, 2008, 75:1180
14. [14] Yao B, Yang H, Liang Q, et al. Anal Chem, 2006, 78:5845
15. [15] Li S, Dong J Y, Guo C G, et al. Talanta, 2013, 116:259
16. [16] Yang C, Zhu G, Zhang L, et al. Electrophoresis, 2004, 25:1729
17. [17] Liang Y, Cong Y Z, Liang Z, et al. Electrophoresis, 2009, 30:4034
18. [18] Wang T T, Ma J F, Zhu G J, et al. J Sep Sci, 2010, 33:3194
19. [19] Yang C, Wang S S, Chang C Y, et al. Anal Chem, 2010, 82:1580
20. [20] Liu R D, Cheddah S, Liu S Q, et al. Anal Chim Acta, 2019, 1048:204
21. [21] Shen Y F, Smith R D. J Micro Sep, 2000, 12:135
22. [22] Cao J Z, Sun W, Gong F F, et al. Chinese Journal of Pharmaceutical Analysis, 2013, 33(12):2092 曹俊姿, 孙雯, 宫菲菲, 等. 药物分析杂志, 2013, 33(12):2092
23. [23] Lee J H, Yeo J, Park H S, et al. Protein Expres Purif, 2013, 87:17
24. [24] Gao P F, Zhao X Y, He M Y, et al. Chinese Journal of Chromatography, 2013, 31(6):537 高培峰, 赵新颖, 贺木易, 等. 色谱, 2013, 31(6):537
25. [25] Nebija D, Noe C, Urban E, et al, Int J Mol Sci, 2014, 15:6399

扫一扫看文章

计量

PDF下载量: 3
文章访问数: 548
HTML全文浏览量: 65

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

English

Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

计量

通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

English

Preparation of capillary isoelectric focusing with monolithic immobilized pH gradient and its application in determination of isoelectric point of proteins

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

计量

文章相关

通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

CCS Chemistry：颜徐州、鲍哲南：你的皮肤可能是一片SPMs