中文摘要：

利用蛋白质组学方法，将大鼠肝微粒体样品进行胰蛋白酶水解；再利用液相色谱．串联质谱法（LCMS／MS），采用多反应监测模式（MRM），通过测定蛋白质水解后产生的特征酶切肽段，实现同时对大鼠肝微粒体内药物代谢酶P450和UGT的绝对定量。本实验首先建立标准工作曲线，对肝微粒体样品中P450和UGT进行定量，在线性范围内，相关系数r〉0．995，线性关系良好，定量限≤10nmol／L；以合成的稳定同位素标记特征肽段作为内标，对UGT1A1进行定量分析。结果表明，同位素标记特征肽段与未标记肽段色谱行为与质谱响应一致，在基质溶液中同位素标记肽段线性关系良好，利用标准曲线法和稳定同位素稀释法测得UGT1A1含量分别为17．30和18．23nmol／g，两种方法所得结果基本一致，但稳定同位素稀释法操作简便，更适用于复杂样品的高通量测定。

英文摘要：

Strategy for absolute quantification of metabolic enzymes in rat liver microsomes was developed by using shotgun-based proteomic approach. Rat liver microsome was digested by trypsin and the specific peptides of drug metabolic enzymes were determined by multiple reaction monitoring （MRM）-based LC-MS/MS. Firstly, standard curves were employed for the quantification of P450 and uridinediphosphate glucuronosyl transferase （ UGT）, which showed good linearity with the r values were all larger than O. 995 and the low limit of quantification was ≤ 10 nmol/L. Secondly, the synthetic isotope labeled specific peptide was served as internal standard for the quantification of UGTIA1. The stable isotope labeled specific peptide showed the same behavior with unlabeled peptide by LC-MS/MS, and the labeled peptide showed good linearity in matrix solution. The content of UGT1A1 was 17.30 nmol/g protein and 18.23 nmol/g protein obtained by standard curve and stable isotope dilution method, respectively. In summary, the results obtained by two methods were consistent, however, the stable isotope dilution method was more convenient than by standard curve, and it was suitable for high throughput determination in complex system.