Method Development And Application For The Absolute Quantification Of Drug Metabolizing Enzymes By Biological Mass Spectrometry

PURPOSEThrough the integration of quantitative proteomics, analytical methods forquantification of drug metabolizing enzymes in rat/human liver microsomes weredeveloped by liquid chromatography-tandem mass spectrometry (LC-MS/MS)approach. The expression levels of drug metabolizing enzymes were obtained bypreparation and determination of rat/human liver microsomes samples. The studyprovides a new approach for drug metabolism enzymes research in drug developmentand clinical medication.METHODThe peptides with specific amino acid sequence were selected through theanalysis of theoretical trypsin cleavage site of CYP2D, CYP2C70, CYP2A1,CYP2D26, UGT2B17, UGT2B2and UGT1A1of rat, after that the peptides weresynthesized by chemical approach. The detection method of specific peptide wasdeveloped by LC-MS/MS through the optimization of mobile phase constitution,gradient elution program, ion source parameter, mass spectrometry parameter andmultiple reaction monitor (MRM) mode. The sample preparation method wasdeveloped by optimization of trypsin percentage, digestion time and solid phaseextraction. The influence of matrix on standard work curve was inspected by synthesisof stable isotope labeled peptide and preparation of liver microsomes matrix solution.The expression levels of drug metabolism enzymes were obtained by determination ofrat liver microsomes.The peptides with specific amino acid sequence were selected through theanalysis of theoretical trypsin cleavage site of CYP1A2, CYP2B6, CYP3A4, CYP3A5,CYP2C9, CYP2C19and CYP2E1of human, and then the stable isotope labeledpeptides were synthesized. The determination method of specific peptides and isotopelabeled peptides was built by LC-MS/MS in MRM mode. The expression levels ofP450isoforms were obtained by determination of12human liver microsomes samples. Bufalin and midazolam were selected as probe substrate and incubated withliver microsomes, after that the CYP3A enzyme activitie was obtained. Correlationanalysis was conducted between enzyme antivity and expression levels of CYP3A4and CYP3A5to inspect the specificity of bufalin and the accuracy of P450isoformscontent.Stable isotope labeled peptides with trypsin cleavage sites were synthesized asinternal standards. The proteolysis process of cleavable peptide was first inspected.The isoforms of CYP2A1, CYP2D26and UGT1A1in rat liver microsomes wereselected as research objects, and the labeled peptides were added to samples by threewayes. The influence of internal peptide on results was inspected, and the proteolysistime and trypsin concentration were inspected simultaneously.RESULTSThe specific peptides of CYP2D, CYP2C70, CYP2A1, CYP2D26, UGT2B17,UGT2B2and UGT1A1of rat were confirmed successively as GNPESSFNDANLR,YIDFVPIPLPR, VHEEIEQVIGR, FADIVPTNIPHMTSR, IILNELAQR,LLDVWTYELPR and SVFDQDPFLLR, the precursor to product ion transitions byMRM mode were selected successively at m/z711.1/625.5,665.6/692.4,437.0/345.2,567.2/577.4,535.5/843.5,702.7/964.5and669.1/645.1, the created standard workcurves were y=820x+1560（r=0.9982）, y=904x–733（r=0.9977）, y=1190x–7210（r=0.9985）, y=1170x–3330（r=0.9993）, y=509x–567（r=0.9996）, y=222x–720（r=0.9981）and y=835x–2420（r=0.9969）, the expression levels ofenzymes were11.77,15.80,26.82,18.30,11.47,27.17and17.30pmol/mg protein. Asynthetic stable isotope labeled peptide was used as internal standard to determine theexpression level of UGTA1. The labeled peptide behaved similarly to the unlabeledpeptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1concentration was detected by the labeled peptide method to be18.2pmol/mg proteincompared with17.3pmol/mg protein obtained by the standard curve method.Although the consistent results were achieved by the two methods, the stable isotopedilution method was more convenient and more suitable for high throughputdeterminations in complex systems.The specific peptides of CYP1A2, CYP2B6, CYP3A4, CYP3A5, CYP2C9,CYP2C19and CYP2E1of human were confirmed successively as YLPNPALQ,FSVTTMR, EVTNFLR, SLGPVGFMK, SHMPYTDAVVHEVQR, HFPLAER, GIIFNNGPTWK, the average expression levels of enzymes were39.3,4.3,54.0,4.6,10.3,3.0and9.3pmol/mg protein. Different P450isoforms had great difference onexpression levels, and the same P450isoform shown significant difference amongindividuals. The expression levels of CYP1A2were slightly higher in smokers, andthe expression levels of other common P450isoforms were slightly lower. Throughthe correlation analysis, the CYP3A4expression level had the best correlation withenzyme activity which was obtained by bufalin as probe. As a substrate of CYP3A4,bufalin had better specificity than midazolam. It also showed that the absolutequantification method for human P450isoforms in the study was accurate andreliable.As the results shown, the cleavable peptide can be digested when three aminoacids were added to each side of the trypsin cleavage site. The accuracy and precisionof results will be increase by selecting cleavable peptides as internal standard,simultenously by selecting suitable enzymolysis time.By the three parts above, the quantification methods for drug metabolismenzymes were developed, and a new approach in drug metabolism enzymes researchwas provided for drug development.