Wang Michael Zhuo, Wu Judy Qiju, Dennison Jennifer B, Bridges Arlene S, Hall Stephen D, Kornbluth Sally, Tidwell Richard R, Smith Philip C, Voyksner Robert D, Paine Mary F, Hall James Edwin
School of Pharmacy, The University of North Carolina at Chapel Hill, NC 27599, USA.
Proteomics. 2008 Oct;8(20):4186-96. doi: 10.1002/pmic.200800144.
The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV <or=27%). The MS-based method strongly correlated with the immunoquantification method (r(2)>or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.
人类细胞色素P450（P450）超家族由膜结合蛋白组成，这些蛋白可代谢多种外源性物质和内源性化合物。在正常和诱导条件下对各种组织中P450表达进行定量，在药物安全性和有效性方面具有重要作用。传统的免疫定量方法动态范围差、通量低且特异性抗体数量有限。基于质谱的定量蛋白质组学的最新进展使得能够在复杂生物混合物中进行绝对蛋白质定量。我们开发了一种基于无胶质谱的蛋白质定量策略，用于定量人肝微粒体（HLM）中的CYP3A酶。分别使用重组蛋白衍生的蛋白型肽和合成的稳定同位素标记蛋白型肽作为校准标准品和内标。定量下限约为20 fmol P450。在两组单独检测的HLM样本（n = 11和n = 22）中，可重复测定CYP3A、CYP3A4和CYP3A5的浓度（CV≤27%）。基于质谱的方法与免疫定量方法（r²≥0.87）和标记物活性（r²≥0.88）密切相关，包括睾酮6β-羟基化（CYP3A）、咪达唑仑1'-羟基化（CYP3A）、伊曲康唑6-羟基化（CYP3A4）和CYP3A5介导的长春新碱M1形成（CYP3A5）。综上所述，我们基于质谱的方法提供了一种特异、灵敏且可靠的P450蛋白定量方法，应有助于在药物开发过程中对P450进行表征，特别是在没有特定底物和/或抗体的情况下。
