Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay
Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay
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结合蛋白质组学和化学探针检测对人肝微粒体中细胞色素 P450 亚型进行定量分析
DOI：
10.1002/pmic.201400025
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发表时间：
2014
期刊：
影响因子：
3.4
通讯作者：
Gu Jingkai
中科院分区：
文献类型：
--
作者：
Liu Xidong;Hu Lianghai;Ge Guangbo;Yang Bo;Ning Jing;Sun Shixin;Yang Ling;Pors Klaus;Gu Jingkai
细胞色素P450(CYP)是最重要的药物代谢酶家族之一，参与多种内源和外源化合物的生物转化。在药物开发中研究新药分子的疗效和评估药物与药物的相互作用时，对CYP表达水平的定量分析是很重要的。目前，尽管序列同源性较高的异构体之间存在交叉反应，但基于化学探针的方法仍是目前应用最广泛的评价CYP活性的方法。因此，为了满足在学术、制药和临床环境中开展药代动力学和个性化药物的日益增长的要求，对每种同工酶的定量是非常必要的。本研究采用蛋白质组学方法，结合稳定同位素稀释法，采用绝对定量的方法分析了七种异构体CYP1A2、2B6、3A4、3A5、2C9、2C19和2E1。从12个人肝微粒体样品中测得1A2、2B6、3A4、3A5、2C9、2C19和2E1的平均绝对量分别为39.3、4.3、54.0、4.6、10.3、3.0和9.3(pmol/mg蛋白)。重要的是，CYP3A4的表达水平与功能活性高度相关(r=0.943，p<；0.0001)，这是用我们开发的一种高选择性的化学探针--蟾酥灵来测量的。MRM鉴定和功能活性分析的结合，如在CYP3A4的情况下，提供了一种可以扩展到其他在药学研究中具有广泛应用的功能酶研究的方法。
Cytochrome P450 (CYP) is one of the most important drug‐metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug–drug interactions in drug development. At present, chemical probe‐based assay is the most widely used approach for the evaluation of CYP activity although there are cross‐reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever‐increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome‐derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r= 0.943,p< 0.0001) with the functional activity, which was measured using bufalin—a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.