Development of an online SPE-LC-MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors.
Development of an online SPE-LC-MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors.
复制标题
使用内源性底物来开发基于SPE-LC-MS的在线基于SPE-LC-MS的测定法，以研究可溶性环氧水解酶（SEH）抑制剂。
DOI：
10.1007/s00216-011-4861-2
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发表时间：
2011-05
影响因子：
4.3
通讯作者：
Hammock, Bruce D.
中科院分区：
文献类型：
--
作者：
Schebb, Nils Helge;Huby, Marion;Morisseau, Christophe;Hwang, Sung Hee;Hammock, Bruce D.
关键词：
可溶性环氧化物水解酶（sEH）是治疗高血压、疼痛和炎症相关疾病的一个有前途的治疗靶点。为了开发sEH抑制剂（sEHIs），需要测定其效价。因此，我们开发了一种新的方法，利用花生四烯酸的环氧化物（14(15)-EpETrE）作为底物。孵育样品未经纯化直接注入在线固相萃取（SPE）液相色谱-电喷雾电离串联质谱（LC-ESI-MS-MS）装置中，整个梯度分离的总运行时间仅为108 s。用湍流色谱法在30 s内从基质中提取分析物。随后，在填充1.7 μm核壳颗粒的50X2.1 mm RP-18柱上进行全梯度分离。采用ESI-MS-MS在SRM模式下对分析物进行了高灵敏度的检测。底物14(15)-EpETrE以96±1 s的稳定保留时间洗脱，其sEH水解产物14,15- dihetre以63±1 s的稳定保留时间洗脱，峰宽窄（最大高度一半的全宽为1.5±0.1 s）。该方法的分析性能优良，柱上检测限为2 fmol，线性范围超过3个数量级，对14,15- dihetre的残留可忽略不计，为0.1%。酶分析以96孔板形式进行，每种抑制剂的12种浓度在22分钟内获得了接近完美的s型剂量-响应曲线，从而可以精确测定IC50值。与其他方法相比，该方法能够定量评估具有皮摩尔电位的强效sEH，因为反应容器中仅使用了33 pmol L−1 sEH。这是通过对10种化合物的活性排序来证明的；荧光法的IC50均≤1 nmol L−1。13种抑制剂的IC50值为>.1 nmol L−1，与荧光法的相关性较好（线性相关系数0.9，斜率0.95,Spearman’s rho 0.9）。然而，对于单个化合物，这种方法和荧光方法之间的效价差异高达8倍。因此，使用天然底物的酶分析，如本文所述，对于可靠地确定sEH抑制的结构-活性关系是必不可少的。本文的在线版本（doi:10.1007/s00216-011-4861-2）包含补充材料，仅供授权用户使用。
Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC50 values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L−1 sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC50 ≤ 1 nmol L−1. Comparison of 13 inhibitors with IC50 values >1 nmol L−1 showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition. The online version of this article (doi:10.1007/s00216-011-4861-2) contains supplementary material, which is available to authorized users.
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影响因子：
7.3
作者：
Rose TE;Morisseau C;Liu JY;Inceoglu B;Jones PD;Sanborn JR;Hammock BD
通讯作者：
Hammock BD
影响因子：
3.1
作者：
Schebb, Nils Helge;Inceoglu, Bora;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
影响因子：
4.1
作者：
Schebb, Nils Helge;Falck, David;Hayen, Heiko
通讯作者：
Hayen, Heiko
影响因子：
7.3
作者：
Hwang, Sung Hee;Tsai, Hsing-Ju;Hammock, Bruce D.
通讯作者：
Hammock, Bruce D.
DOI：
10.1016/s1570-0232(02)00914-5
发表时间：
2003-03-05
影响因子：
3
作者：
Polson, C;Sarkar, P;Grant, R
通讯作者：
Grant, R