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50μL,将终浓度为20 nmol/L的TPK2与终浓度为5μmol/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1μmol/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.
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 C^s column within 4 min, and quantified by electrospray ionization mass spectrometry（ESI-MS）. An online desalting pro- cedure 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.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2013年第5期1111-1114,共4页
Chemical Journal of Chinese Universities
基金
国家自然科学基金(批准号:2011BAI03B01)
吉林省医药产业发展专项资金(批准号:YYZX201131)资助
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
Alzheimer＇ s disease
Tau protein kinase
Rapid resolution liquid chromatography/mass spec-trometry
