Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny
Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny
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DOI：
10.1124/dmd.115.067538
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发表时间：
2016-07-01
影响因子：
3.9
通讯作者：
Wang, Michael Zhuo
中科院分区：
文献类型：
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作者：
Chen, Yao;Zane, Nicole R.;Wang, Michael Zhuo
含黄素单加氧酶（FMO）在小分子药物的代谢中具有重要作用。在五种人类FMO中，FMO1、FMO3和FMO5与肝脏药物代谢最相关。尽管之前已报道过基于免疫定量的 FMO1 和 FMO3 的年龄依赖性肝蛋白表达，但有关肝 FMO5 蛋白表达的信息很少。为了克服免疫定量的局限性，开发并优化了一种基于超高效液相色谱 (UPLC) 多反应监测 (MRM) 的靶向定量蛋白质组学方法，用于定量人肝微粒体 (HLM) 中的 FMO1、FMO3 和 FMO5。纳入了硅后产物离子筛选过程，以在合成前验证潜在特征肽的 LC-MRM 检测。通过关联一组成年个体供体 HLM（年龄 39-67 岁）中的标记物底物活性和蛋白质表达，对所开发的方法进行了验证。 FMO3和FMO5的平均（范围）蛋白表达分别为46（26-65）pmol/mg HLM蛋白和27（11.549）pmol/mgHLM蛋白。为了证明 FMO1 的定量，对一组胎儿个体供体 HLM（胎龄 14-20 周）进行了分析。 FMO1蛋白表达的平均（范围）为7.0（4.9-9.7）pmol/mg HLM蛋白。此外，在胎儿、儿童和成人 HLM 中评估了 FMO5 的个体发育蛋白表达。还使用两种不同的校准标准（重组蛋白与合成特征肽）对 FMO 蛋白的定量进行比较，以评估全蛋白与总蛋白之间的比率。总之，我们开发了一种基于 UPLC-MRM 的靶向定量蛋白质组学方法，用于 HLM 中 FMO 酶的定量。
Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developedmethodwas validated by correlatingmarker substrate activity and protein expression in a panel of adult individual donor HLM(age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.549) pmol/mgHLMprotein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. Themean (range) FMO1 protein expressionwas 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM.