AB SCIEX, Framingham, MA 01701, USA.
Proteomics. 2011 Jan;11(1):33-41. doi: 10.1002/pmic.201000456. Epub 2010 Dec 6.
The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.
细胞色素 P450（CYP）蛋白是一组膜结合蛋白，作为人体内主要的代谢酶发挥作用。CYP 诱导的定量分析对于确定药物在人体内的处置、安全性和疗效至关重要。本文描述了一种无胶、高通量的 LC-MS 方法，通过测量肝细胞孵育物中酶解释放的同工型特异性肽来定量 CYP 同工型 1A2、2B6、3A4 和 3A5。该方法使用合成的稳定同位素标记肽作为内标，允许从肝微粒体制剂中进行相对和绝对定量。通过该 LC-MS 方法测定的 CYP 蛋白与 CYP1A2、CYP2B6 和 CYP3A4 诱导水平的 mRNA 和活性相关性良好。有趣的是，苯巴比妥诱导 3A5 的倍数变化较小。对于样品的重复分析，该方法的结果具有良好的重现性，平均 CV 小于 10%。该 LC-MS 方法为 CYP 蛋白定量提供了一种稳健的测定方法，可用于 CYP 诱导测定。
