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Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples

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Abstract

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.

Calibration curve in complex matrix for CYPs quantification

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References

  1. Guengerich FP (2004) Cytochrome P450: what have we learned and what are the future issues? Drug Metab Rev 36:159–197

    Article  CAS  Google Scholar 

  2. Ortiz de Montellano PR (ed) (2005) Cytochrome P450: structure, mechanism, and biochemistry. Kluwer/Plenum, New York

    Google Scholar 

  3. Lewis DF (2004) 57 varieties: the human cytochromes P450. Pharmacogenomics 5:305–318

    Article  CAS  Google Scholar 

  4. Zanger UM, Schwab M (2013) Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 138:103–141

    Article  CAS  Google Scholar 

  5. Nishimura M, Yaguti H, Yoshitsugu H, Naito S, Satoh T (2003) Tissue distribution of mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 123:369–375

    Article  CAS  Google Scholar 

  6. Bièche I, Narjoz C, Asselah T, Vacher S, Marcellin P, Lidereau R, Beaune P, de Waziers I (2007) Reverse transcriptase-PCR quantification of mRNA levels from cytochrome (CYP)1, CYP2 and CYP3 families in 22 different human tissues. Pharmacogenet Genomics 17:731–742

    Article  Google Scholar 

  7. Huber M, Bahr I, Krätzschmar JR, Becker A, Müller E-C, Donner P, Pohlenz H-D, Schneider MR, Sommer A (2004) Comparison of proteomic and genomic analyses of the human breast cancer cell line T47D and the antiestrogen-resistant derivative T47D-r. Mol Cell Proteomics 3:43–55

    Article  CAS  Google Scholar 

  8. Williamson BL, Purkayastha S, Hunter CL, Nuwaysir L, Hill J, Easterwood L, Hill J (2011) Quantitative protein determination for CYP induction via LC-MS/MS. Proteomics 11:33–41

    Article  CAS  Google Scholar 

  9. Snawder JE, Lipscomb JC (2000) Interindividual variance of cytochrome P450 forms in human hepatic microsomes: correlation of individual forms with xenobiotic metabolism and implications in risk assessment. Regul Toxicol Pharmacol 32:200–209

    Article  CAS  Google Scholar 

  10. De Bock L, Colin P, Boussery K, Van Bocxlaer J (2012) Development and validation of an enzyme-linked immunosorbent assay for the quantification of cytochrome 3A4 in human liver microsomes. Talanta 99:357–362

    Article  Google Scholar 

  11. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423

    CAS  Google Scholar 

  12. Paine MF (2006) The human intestinal cytochrome P450 “PIE”. Drug Metab Dispos 34:880–886

    Article  CAS  Google Scholar 

  13. de Hoffmann E, Stroobant V (2007) Mass spectrometry: principles and applications. Wiley, Chichester

    Google Scholar 

  14. Jenkins RE, Kitteringham NR, Hunter CL, Webb S, Hunt TJ, Elsby R, Watson RB, Williams D, Pennington SR, Park BK (2006) Relative and absolute quantitative expression profiling of cytochromes P450 using isotope-coded affinity tags. Proteomics 6:1934–1947

    Article  CAS  Google Scholar 

  15. Duan X, Chen X, Yang Y, Zhong D (2007) Precolumn derivatization of cysteine residues for quantitative analysis of five major cytochrome P450 isoenzymes by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 21:3234–3244

    Article  CAS  Google Scholar 

  16. Wang Y, Al-Gazzar A, Seibert C, Sharif A, Lane C, Griffiths WJ (2006) Proteomic analysis of cytochromes P450: a mass spectrometry approach. Biochem Soc Trans 34:1246–1251

    Article  CAS  Google Scholar 

  17. Huang H-J, Tsai M-L, Chen Y-W, Chen S-H (2011) Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome. J Proteome 74:2734–2744

    Article  CAS  Google Scholar 

  18. Jia N, Liu X, Wen J, Qian L, Qian X, Wu Y, Fan G (2007) A proteomic method for analysis of CYP450s protein expression changes in carbon tetrachloride induced male rat liver microsomes. Toxicology 237:1–11

    Article  CAS  Google Scholar 

  19. Lane CS, Wang Y, Betts R, Griffiths WJ, Patterson LH (2007) Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling. Mol Cell Proteomics 6:953–962

    Article  CAS  Google Scholar 

  20. Wang MZ, Wu JQ, Dennison JB, Bridges AS, Hall SD, Kornbluth S, Tidwell RR, Smith PC, Voyksner RD, Paine MF et al (2008) A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes. Proteomics 8:4186–4196

    Article  CAS  Google Scholar 

  21. Langenfeld E, Zanger UM, Jung K, Meyer HE, Marcus K (2009) Mass spectrometry-based absolute quantification of microsomal cytochrome P450 2D6 in human liver. Proteomics 9:2313–2323

    Article  CAS  Google Scholar 

  22. Seibert C, Davidson BR, Fuller BJ, Patterson LH, Griffiths WJ, Wang Y (2009) Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry. J Proteome Res 8:1672–1681

    Article  CAS  Google Scholar 

  23. Kawakami H, Ohtsuki S, Kamiie J, Suzuki T, Abe T, Terasaki T (2011) Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. J Pharm Sci 100:341–352

    Article  CAS  Google Scholar 

  24. Ohtsuki S, Schaefer O, Kawakami H, Inoue T, Liehner S, Saito A, Ishiguro N, Kishimoto W, Ludwig-Schwellinger E, Ebner T, Terasaki T (2012) Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities. Drug Metab Dispos 40:83–92

    Article  CAS  Google Scholar 

  25. Shawahna R, Uchida Y, Declèves X, Ohtsuki S, Yousif S, Dauchy S, Jacob A, Chassoux F, Daumas-Duport C, Couraud P-O, Terasaki T, Scherrmann J-M (2011) Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Mol Pharm 8:1332–1341

    Article  CAS  Google Scholar 

  26. Sato Y, Miyashita A, Iwatsubo T, Usui T (2012a) Simultaneous absolute protein quantification of carboxylesterases 1 and 2 in human liver tissue fractions using liquid chromatography-tandem mass spectrometry. Drug Metab Dispos 40: 1389–1396

  27. Sato Y, Nagata M, Kawamura A, Miyashita A, Usui T (2012) Protein quantification of UDP-glucuronosyltransferases 1A1 and 2B7 in human liver microsomes by LC-MS/MS and correlation with glucuronidation activities. Xenobiotica 42: 823–829

  28. Liao W-L, Heo G-Y, Dodder NG, Pikuleva IA, Turko IV (2010) Optimizing the conditions of a multiple reaction monitoring assay for membrane proteins: quantification of cytochrome P450 11A1 and adrenodoxin reductase in bovine adrenal cortex and retina. Anal Chem 82:5760–5767

    Article  CAS  Google Scholar 

  29. Achour B, Russell MR, Barber J, Rostami-Hodjegan A (2014) Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics. Drug Metab Dispos 42:500–510

    Article  CAS  Google Scholar 

  30. Zhang H, Liu Q, Zimmerman LJ, Ham A-JL, Slebos RJC, Rahman J, Kikuchi T, Massion PP, Carbone DP, Billheimer D et al (2011) Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry. Mol Cell Proteomics 10:M110.006593

    Article  Google Scholar 

  31. Pailleux F, Beaudry F (2012) Internal standard strategies for relative and absolute quantitation of peptides in biological matrices by liquid chromatography tandem mass spectrometry. Biomed Chromatogr 26:881–891

    CAS  Google Scholar 

  32. Yu A-M, Qu J, Felmlee MA, Cao J, Jiang X-L (2009) Quantitation of human cytochrome P450 2D6 protein with immunoblot and mass spectrometry analysis. Drug Metab Dispos Biol Fate Chem 37:170–177

    Article  CAS  Google Scholar 

  33. Sun L, Zhang Y, Tao D, Zhu G, Zhao Q, Wu Q, Liang Z, Yang L, Zhang L, Zhang Y (2012) SDS-PAGE-free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes. Proteomics 12:3464–3469

    Article  CAS  Google Scholar 

  34. Shrivas K, Mindaye ST, Getie-Kebtie M, Alterman MA (2013) Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450. Toxicol Appl Pharmacol 267:125–136

    Article  CAS  Google Scholar 

  35. Roos PH, Venkatachalam A, Manz A, Waentig L, Koehler CU, Jakubowski N (2008) Detection of electrophoretically separated cytochromes P450 by element-labelled monoclonal antibodies via laser ablation inductively coupled plasma mass spectrometry. Anal Bioanal Chem 392:1135–1147

    Article  CAS  Google Scholar 

  36. Alterman MA, Kornilayev B, Duzhak T, Yakovlev D (2005) Quantitative analysis of cytochrome P450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach. Drug Metab Dispos Biol Fate Chem 33:1399–1407

    Article  CAS  Google Scholar 

  37. Kremers P, Beaune P, Cresteil T, De Graeve J, Columelli S, Leroux J-P, Gielen JE (1981) Cytochrome P-450 monooxygenase activities in human and rat liver microsomes. Eur J Biochem 118:599–606

    Article  CAS  Google Scholar 

  38. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B (1998) A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95:2509–2514

    Article  CAS  Google Scholar 

  39. Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, Sekine Y, Uchida Y, Ito S, Terasaki T (2008) Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res 25:1469–1483

    Article  CAS  Google Scholar 

  40. Yang Z, Attygalle AB (2007) LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides. J Mass Spectrom 42:233–243

    Article  CAS  Google Scholar 

  41. Violette A, Biass D, Dutertre S, Koua D, Piquemal D, Pierrat F, Stöcklin R, Favreau P (2012) Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach. J Proteome 75:5215–5225

    Article  CAS  Google Scholar 

  42. Boja ES, Fales HM (2001) Overalkylation of a protein digest with iodoacetamide. Anal Chem 73:3576–3582

    Article  CAS  Google Scholar 

  43. Klein K, Winter S, Turpeinen M, Schwab M, Zanger UM (2010) Pathway-targeted pharmacogenomics of CYP1A2 in human liver. Front Pharmacol. doi:10.3389/fphar.2010.00129

    Google Scholar 

  44. Rowland Yeo K, Rostami-Hodjegan A, Tucker GT (2004) Abundance of cytochromes P450 in human liver: a meta-analysis. Br J Clin Pharmacol 57:687–688

    Google Scholar 

  45. Guengerich FP, Turvy CG (1991) Comparison of levels of several human microsomal cytochrome P-450 enzymes and epoxide hydrolase in normal and disease states using immunochemical analysis of surgical liver samples. J Pharmacol Exp Ther 256:1189–1194

    CAS  Google Scholar 

  46. Rodrigues AD (1999) Integrated cytochrome P450 reaction phenotyping. Biochem Pharmacol 57:465–480

    Article  CAS  Google Scholar 

  47. Stevens JC, Marsh SA, Zaya MJ, Regina KJ, Divakaran K, Le M, Hines RN (2008) Developmental changes in human liver CYP2D6 expression. Drug Metab Dispos 36:1587–1593

    Article  CAS  Google Scholar 

  48. Dennison JB, Jones DR, Renbarger JL, Hall SD (2007) Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes. J Pharmacol Exp Ther 321:553–563

    Article  CAS  Google Scholar 

  49. Gaedigk A, Baker DW, Totah RA, Gaedigk R, Pearce RE, Vyhlidal CA, Zeldin DC, Leeder JS (2006) Variability of CYP2J2 expression in human fetal tissues. J Pharmacol Exp Ther 319:523–532

    Article  CAS  Google Scholar 

  50. Yamazaki H, Okayama A, Imai N, Guengerich FP, Shimizu M (2006) Inter-individual variation of cytochrome P4502J2 expression and catalytic activities in liver microsomes from Japanese and Caucasian populations. Xenobiotica 36:1201–1209

    Article  CAS  Google Scholar 

  51. Xu M, Ju W, Hao H, Wang G, Li P (2013) Cytochrome P450 2 J2: distribution, function, regulation, genetic polymorphisms and clinical significance. Drug Metab Rev 45:311–352

    Article  CAS  Google Scholar 

  52. Avadhani NG, Sangar MC, Bansal S, Bajpai P (2011) Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals. FEBS J 278:4218–4229

    Article  CAS  Google Scholar 

  53. Knockaert L, Fromenty B, Robin M-A (2011) Mechanisms of mitochondrial targeting of cytochrome P450 2E1: physiopathological role in liver injury and obesity. FEBS J 278:4252–4260

    Article  CAS  Google Scholar 

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Acknowledgment

Financial support of the Institut de Chimie des Substances Naturelles (CNRS-ICSN) is gratefully acknowledged.

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Authors and Affiliations

  1. Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198, Gif-sur-Yvette Cedex, France

    Ahmad Al Ali, David Touboul, Jean-Pierre Le Caër, Isabelle Schmitz-Afonso & Alain Brunelle

  2. Médecine Personnalisée, Pharmacogénomique et Optimisation Thérapeutique, INSERM UMR-S1147, Centre Universitaire des Saints-Pères, Université Paris Descartes, Sorbonne Paris-Cité, 45 rue des Saint Pères, 75270, Paris Cedex 06, France

    Ahmad Al Ali, Jean-Pierre Flinois, Catherine Marchetti, Isabelle De Waziers & Philippe Beaune

  3. Chimie-Toxicologie Analytique et Cellulaire, UMR CNRS 8638, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, 4 avenue de l’Observatoire, 75006, Paris, France

    Olivier Laprévote

  4. Service de Toxicologie Biologique, Hôpital Lariboisière, 2 rue Ambroise Paré, 75010, Paris, France

    Olivier Laprévote

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  1. Ahmad Al Ali
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  2. David Touboul
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Correspondence to David Touboul.

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Al Ali, A., Touboul, D., Le Caër, JP. et al. Optimization and validation of a label-free MRM method for the quantification of cytochrome P450 isoforms in biological samples. Anal Bioanal Chem 406, 4861–4874 (2014). https://doi.org/10.1007/s00216-014-7928-z

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  • DOI: https://doi.org/10.1007/s00216-014-7928-z

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