Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry
Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry
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毛细管电泳结合高效液相色谱-串联质谱法筛选天然产物提取物中雷帕霉素抑制剂的哺乳动物靶点
DOI：
10.1016/j.chroma.2015.02.033
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发表时间：
2015
影响因子：
4.1
通讯作者：
Kang Jingwu
中科院分区：
文献类型：
--
作者：
Zhang Yanmei;Li Feng;Li Mingxia;Kang Jingwu
本研究采用毛细管电泳（CE）结合HPLC-MS/MS技术，从天然产物提取物中筛选雷帕霉素靶蛋白（mTOR）抑制剂。利用5-羧基荧光素标记的底物肽F-4 EBP 1、已知的mTOR抑制剂AZD 8055和由18种天然产物提取物组成的小型化学文库建立了筛选系统。用毛细管电泳-激光诱导荧光检测法对天然产物提取物进行生化筛选。CE分离允许定量测量磷酸化产物，因此定量酶抑制以及抑制动力学。只要磷酸化产物的峰面积与阴性对照相比降低，就很容易识别命中。活性天然产物提取物的后续测定引导分离用HPLC-MS/MS进行以追踪特定活性组分。通过MS/MS分析提供的分子离子和碎片信息，对所鉴定的活性成分进行了结构鉴定。基于CE的测定方法仅需要微量纯化合物，其可以容易地通过HPLC纯化。因此，毛细管电泳和高效液相色谱-串联质谱的结合为从天然粗提物中筛选生物活性化合物提供了一个高通量的平台。利用该筛选系统，从丹参提取物中发现了新的mTOR抑制剂丹参素A和丹参素C。
In this study, capillary electrophoresis (CE) combined with HPLC–MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC–MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentaion information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC–MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.