高等学校化学学报

高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (5): 1111.doi: 10.7503/cjcu20120786

• 分析化学 • 上一篇    下一篇

快速高分离度液相色谱-质谱联用方法筛选Tau蛋白激酶2抑制剂

陈宁1,2, 孙一3, 越皓3, 刘淑莹1,3   

  1. 1. 中国科学院长春应用化学研究所, 长春质谱中心, 长春 130022;
    2. 中国科学院大学, 北京100049;
    3. 长春中医药大学, 吉林省人参科学研究院, 长春 130117
  • 收稿日期:2012-08-28 出版日期:2013-05-10 发布日期:2013-05-10
  • 通讯作者: 刘淑莹, 女, 博士, 研究员, 博士生导师, 主要从事生物质谱方面的研究. E-mail: syliu19@yahoo.com.cn E-mail:syliu19@yahoo.com.cn
  • 基金资助:

    国家自然科学基金(批准号: 2011BAI03B01)和吉林省医药产业发展专项资金(批准号: YYZX201131)资助.

Screening the Inhibitors of Tau Protein Kinase 2 by Rapid Resolution Liquid Chromatogrophy-Mass Spectrometry

CHEN Ning1,2, SUN Yi3, YUE Hao3, LIU Shu-Ying1,3   

  1. 1. Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China;
    2. University of Chinese Academy of Sciences, Beijing 100049, China;
    3. Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China
  • Received:2012-08-28 Online:2013-05-10 Published:2013-05-10

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被引次数

3

摘要：

建立了测定Tau 蛋白激酶2(Tau protein kinase 2, TPK2)催化反应产物磷酸化十肽(PKpTPKKAKKL)的快速高分离度液相色谱-质谱方法(RRLC/MS)用于筛选TPK2抑制剂. 反应溶液总体积为50 μL, 将终浓度为20 nmol/L的TPK2与终浓度为5 μmol/L的底物十肽(PKTPKKAKKL)于30 ℃反应30 min, 用等体积乙腈终止反应并加入终浓度为1 μmol/L的磷酸化十肽(PKpTPKKAKKV)作为内标, 采用Agilent SB-C18色谱柱, 以乙腈-水为流动相梯度洗脱分离后, 用RRLC/MS进行定性及定量分析. 该方法可通过精确定量一定时间内的酶促反应产物来反映酶活性被抑制的程度, 作为TPK2抑制剂的筛选方法, 本方法具有简单、 灵敏、 快速的优点, 能够避免光谱法筛选可能产生的假阳性结果, 可用于TPK2的先导化合物的高通量筛选.

关键词: 阿尔兹海默病, Tau蛋白激酶, 快速高分离度液相色谱-质谱

Abstract:

A method based on rapid resolution liquid chromatogrophy-mass spectrometry was developed for screening the inhibitor of Tau protein kinase 2(TPK2) from natural products. Tau protein kinase 2(TPK2) is a serine/threonine kinase, which is reported to play an important role in the pathogenesis of Alzheimer's disease. The excellent substrate, histone-based peptide PKTPKKAKKL was used in the study. The product(PKpTPKKAKKL) and the internal standard(PKpTPKKAKKV) were separated by RRLC on a C18 column within 4 min, and quantified by electrospray ionization mass spectrometry(ESI-MS). An online desalting procedure was carried out in the experiment without sample pretreatment prior to analysis. Moreover, the enzymatic reaction conditions including pH value, temperature, reaction time, the concentration of kinase were optimized.

Key words: Alzheimer's disease, Tau protein kinase, Rapid resolution liquid chromatography/mass spectrometry

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