Liu Xidong, Hu Lianghai, Ge Guangbo, Yang Bo, Ning Jing, Sun Shixin, Yang Ling, Pors Klaus, Gu Jingkai
Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, P.R. China; Research Center for Drug Metabolism, School of Life Sciences, Jilin University, Changchun, P.R. China.
Proteomics. 2014 Aug;14(16):1943-51. doi: 10.1002/pmic.201400025. Epub 2014 Jul 14.
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.
细胞色素P450（CYP）是最重要的药物代谢酶家族之一，参与许多内源性和外源性化合物的生物转化。在研究新药分子的疗效以及评估药物开发中的药物相互作用时，对CYP表达水平进行定量分析非常重要。目前，基于化学探针的测定法是评估CYP活性最广泛使用的方法，尽管具有高序列同源性的同工型之间存在交叉反应。因此，为了满足学术、制药和临床环境中对进行药代动力学和个性化医疗日益增长的需求，非常需要对每种同工酶进行定量。在此，采用绝对定量方法，结合稳定同位素稀释分析，使用蛋白质组学方法分析CYP1A2、2B6、3A4、3A5、2C9、2C19和2E1这七种同工型。从十二个人类肝微粒体样品中测得的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蛋白质）。重要的是，CYP3A4的表达水平与功能活性显示出高度相关性（r = 0.943，p < 0.0001），功能活性是使用我们开发的高选择性化学探针蟾毒灵进行测量的。如CYP3A4的情况那样，MRM鉴定与功能活性分析的结合提供了一种方案，该方案可扩展到其他功能酶研究，并在药物研究中具有广泛应用。
