期刊
DRUG METABOLISM AND DISPOSITION
卷 44, 期 7, 页码 975-983出版社
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/dmd.115.067538
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资金
- National Institutes of Health National Institute of General Medical Sciences [R01GM089994]
- Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health [5 T32 GM086330-04]
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.
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