基于酪氨酸电化学的蛋白质磷酸化检测
杨郁，郭良宏*，曲娜，韦明元
中国科学院生态环境研究中心，环境化学与生态毒理学国家重点实验室，
北京市海淀区双清路18号，邮编100085
*联系人：电话：010-62849685，E-mail地址：
蛋白质磷酸化是蛋白质翻译后修饰中最常见、最重要的一种共价修饰方式，在许多生物效应中起着开/关的作用，是基因表达和蛋白质合成的重要调控者，它几乎参与生命活动的所有过程。尤其在细胞应答外界刺激的信号传递途径中，蛋白质磷酸化是目前所知道的最主要方式。其中负责磷酸化的蛋白激酶的活性常会受到小分子化合物的调控。目前已有文献报道了一些分析方法应用于蛋白质磷酸化的测定，如：[32P]放射性标记法、免疫印迹法、质谱法、荧光法、表面等离子体共振等，这些方法操作繁琐且成本较昂贵。因此，需要建立一种简单、快速、高灵敏的蛋白质磷酸化分析检测方法，并在此基础上研究小分子化合物对蛋白质激酶活性的调控效应。本研究建立了一种免标记的蛋白质磷酸化电化学传感检测方法。通过在氧化铟锡（ITO）电极表面共价固定含酪氨酸的多肽分子，该分子为蛋白质酪氨酸激酶的底物。以底物分子中的酪氨酸作为电化学信号探针，通过电子媒介体催化放大酪氨酸的电化学信号。利用正常酪氨酸和磷酸化酪氨酸之间电催化信号的差异，实现对蛋白质磷酸化反应的检测，并以此来研究小分子化合物对蛋白质酪氨酸激酶的调控效应。本研究中重点解决了电子媒介体在修饰电极上的电化学动力学、多肽分子固定的表面化学、激酶催化的多肽磷酸化反应的电化学检测机制等关键问题。
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 90813016, 21077124 and 20907060).
References
[1] Ahn, N. Chem. Rev. 2001, 101, 2207-2207.
[2] Johnson, L. N.; Lewis, R. J. Chem. Rev. 2001, 101, 2209-2242.
[3] Adams, J. A. Chem. Rev. 2001, 101, 2271-2290.
[4] Goldsmith, E. J.; Akella, R.; Min, X. S.; Zhou, T. J.; Humphreys, J. M. Chem. Rev. 2007, 107, 5065-5081.
[5] Burkhard, K.; Smith, S.; Deshmukh, R.; MacKerell, A. D.; Shapiro, P. Curr. Top. Med. Chem. 2009, 9, 678-689.
[6] Li, T.; Liu, D. J.; Wang, Z. X. Anal. Chem. 2010, 82, 3067-3072.
Label-Free Electrochemical Measurement of Protein Tyrosine Kinase Activity and Inhibition Based on Electro-catalyzed
Tyrosine Signaling
Yu Yang, Liang-Hong Guo*, Na Qu, Ming-Yuan Wei
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18
Shuangqing Road, P.O.Box 2871, Beijing 100085, China.
*Corresponding author. Tel. +86 10 62849685; Fax +86 10 62849685.
E-mail address: [email protected]
Abstract
Protein phosphorylation catalyzed by protein kinases plays vital regulatory roles in many metabolic and cell signaling pathways [1-4]. Measurement of the kinase activity and identification of their potential inhibitors are necessary for the understanding of many fundamental biochemical processes and for antitumor drug discovery [5, 6]. In this report, a novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, a EGFR substrate, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)32+ (bpy=2, 2’-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 2 UmL-1. Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC50 of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants Ki were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.
基于酪氨酸电化学的蛋白质磷酸化检测
作者：
作者单位：杨郁， 郭良宏， 曲娜， 韦明元中国科学院生态环境研究中心,环境化学与生态毒理学国家重点实验室,北京市海淀区双清路18号,100085
本文链接：http://d.g.wanfangdata.com.cn/Conference_7488973.aspx
基于酪氨酸电化学的蛋白质磷酸化检测
杨郁，郭良宏*，曲娜，韦明元
中国科学院生态环境研究中心，环境化学与生态毒理学国家重点实验室，
北京市海淀区双清路18号，邮编100085
*联系人：电话：010-62849685，E-mail地址：
蛋白质磷酸化是蛋白质翻译后修饰中最常见、最重要的一种共价修饰方式，在许多生物效应中起着开/关的作用，是基因表达和蛋白质合成的重要调控者，它几乎参与生命活动的所有过程。尤其在细胞应答外界刺激的信号传递途径中，蛋白质磷酸化是目前所知道的最主要方式。其中负责磷酸化的蛋白激酶的活性常会受到小分子化合物的调控。目前已有文献报道了一些分析方法应用于蛋白质磷酸化的测定，如：[32P]放射性标记法、免疫印迹法、质谱法、荧光法、表面等离子体共振等，这些方法操作繁琐且成本较昂贵。因此，需要建立一种简单、快速、高灵敏的蛋白质磷酸化分析检测方法，并在此基础上研究小分子化合物对蛋白质激酶活性的调控效应。本研究建立了一种免标记的蛋白质磷酸化电化学传感检测方法。通过在氧化铟锡（ITO）电极表面共价固定含酪氨酸的多肽分子，该分子为蛋白质酪氨酸激酶的底物。以底物分子中的酪氨酸作为电化学信号探针，通过电子媒介体催化放大酪氨酸的电化学信号。利用正常酪氨酸和磷酸化酪氨酸之间电催化信号的差异，实现对蛋白质磷酸化反应的检测，并以此来研究小分子化合物对蛋白质酪氨酸激酶的调控效应。本研究中重点解决了电子媒介体在修饰电极上的电化学动力学、多肽分子固定的表面化学、激酶催化的多肽磷酸化反应的电化学检测机制等关键问题。
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 90813016, 21077124 and 20907060).
References
[1] Ahn, N. Chem. Rev. 2001, 101, 2207-2207.
[2] Johnson, L. N.; Lewis, R. J. Chem. Rev. 2001, 101, 2209-2242.
[3] Adams, J. A. Chem. Rev. 2001, 101, 2271-2290.
[4] Goldsmith, E. J.; Akella, R.; Min, X. S.; Zhou, T. J.; Humphreys, J. M. Chem. Rev. 2007, 107, 5065-5081.
[5] Burkhard, K.; Smith, S.; Deshmukh, R.; MacKerell, A. D.; Shapiro, P. Curr. Top. Med. Chem. 2009, 9, 678-689.
[6] Li, T.; Liu, D. J.; Wang, Z. X. Anal. Chem. 2010, 82, 3067-3072.
Label-Free Electrochemical Measurement of Protein Tyrosine Kinase Activity and Inhibition Based on Electro-catalyzed
Tyrosine Signaling
Yu Yang, Liang-Hong Guo*, Na Qu, Ming-Yuan Wei
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18
Shuangqing Road, P.O.Box 2871, Beijing 100085, China.
*Corresponding author. Tel. +86 10 62849685; Fax +86 10 62849685.
E-mail address: [email protected]
Abstract
Protein phosphorylation catalyzed by protein kinases plays vital regulatory roles in many metabolic and cell signaling pathways [1-4]. Measurement of the kinase activity and identification of their potential inhibitors are necessary for the understanding of many fundamental biochemical processes and for antitumor drug discovery [5, 6]. In this report, a novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, a EGFR substrate, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)32+ (bpy=2, 2’-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 2 UmL-1. Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC50 of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants Ki were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.
基于酪氨酸电化学的蛋白质磷酸化检测
作者：
作者单位：杨郁， 郭良宏， 曲娜， 韦明元中国科学院生态环境研究中心,环境化学与生态毒理学国家重点实验室,北京市海淀区双清路18号,100085
本文链接：http://d.g.wanfangdata.com.cn/Conference_7488973.aspx