RT Journal Article SR Electronic T1 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 JF Drug Metabolism and Disposition JO Drug Metab Dispos FD American Society for Pharmacology and Experimental Therapeutics SP dmd.115.067538 DO 10.1124/dmd.115.067538 A1 Yao Chen A1 Nicole R Zane A1 Dhiren R Thakker A1 Michael Zhuo Wang YR 2016 UL http://dmd.aspetjournals.org/content/early/2016/02/01/dmd.115.067538.abstract AB 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, a UPLC-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 prior to their synthesis. The developed method was validated by correlating marker 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 (11.5 - 49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9 - 9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins vs. synthetic signature peptides, to assess the ratio between holoprotein vs. total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM.