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A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferase enzymes in the human liver


Cytochrome P450 (CYP450) and 5′-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma–optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2 fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples.

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  1. 1.

    Wang L, Collins C, Kelly EJ, Chu X, Ray AS, Salphati L, et al. Transporter expression in liver tissue from subjects with alcoholic or hepatitis C cirrhosis quantified by targeted quantitative proteomics. Drug Metab Dispos. 2016;44(11):1752–8. https://doi.org/10.1124/dmd.116.071050.

  2. 2.

    Issa NT, Wathieu H, Ojo A, Byers SW, Dakshanamurthy S. Drug metabolism in preclinical drug development: a survey of the discovery process, toxicology, and computational tools. Curr Drug Metab. 2017;18(6):556–65. https://doi.org/10.2174/1389200218666170316093301.

  3. 3.

    Shrestha R, Cho P, Paudel S, Shrestha A, Kang M, Jeong T, et al. Exploring the metabolism of loxoprofen in liver microsomes: the role of cytochrome P450 and UDP-glucuronosyltransferase in its biotransformation. Pharmaceutics. 2018;10(3):112. https://doi.org/10.3390/pharmaceutics10030112.

  4. 4.

    Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Therapeut. 2013;138(1):103–41. https://doi.org/10.1007/s11426-013-5049-8.

  5. 5.

    Prasad B, Vrana M, Mehrotra A, Johnson K, Bhatt DK. The promises of quantitative proteomics in precision medicine. J Pharm Sci-US. 2017;106(3):738–44. https://doi.org/10.1016/j.xphs.2016.11.017.

  6. 6.

    Nakamura K, Hirayama-Kurogi M, Ito S, Kuno T, Yoneyama T, Obuchi W, et al. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: comparison with MRM/SRM and HR-MRM/PRM. Proteomics. 2016;16(15–16):2106–17. https://doi.org/10.1002/pmic.201500433.

  7. 7.

    Vildhede A, Kimoto E, Rodrigues AD, Varma MV. Quantification of hepatic organic anion transport proteins OAT2 and OAT7 in human liver tissue and primary hepatocytes. Mol Pharm. 2018;15(8):3227–35. https://doi.org/10.1021/acs.molpharmaceut.8b00320.

  8. 8.

    Melillo N, Darwich AS, Magni P, Rostami-Hodjegan A. Accounting for inter-correlation between enzyme abundance: a simulation study to assess implications on global sensitivity analysis within physiologically-based pharmacokinetics. J Pharmacokinet Pharmacodyn. 2019;46(2):137–54. https://doi.org/10.1007/s10928-019-09627-6.

  9. 9.

    Aebersold R, Burlingame AL, Bradshaw RA. Western blots versus selected reaction monitoring assays: time to turn the tables? ASBMB. 2013. https://doi.org/10.1074/mcp.E113.031658.

  10. 10.

    Kumar V, Salphati L, Hop CE, Xiao G, Lai Y, Mathias A, et al. A comparison of total and plasma membrane abundance of transporters in suspended, plated, Sandwich-cultured human hepatocytes versus human liver tissue using quantitative targeted proteomics and cell surface biotinylation. Drug Metab Dispos. 2019;47(4):350–7. https://doi.org/10.1124/dmd.118.084988.

  11. 11.

    Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A. 2003;100(12):6940–5. https://doi.org/10.1073/pnas.0832254100.

  12. 12.

    Beynon RJ, Doherty MK, Pratt JM, Gaskell SJ. Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat Methods. 2005;2(8):587. https://doi.org/10.1038/nmeth774.

  13. 13.

    Zhou Y, Shan Y, Zhang L, Zhang Y. Recent advances in stable isotope labeling based techniques for proteome relative quantification. J Chromatogr A. 2014;1365:1–11. https://doi.org/10.1016/j.chroma.2014.08.098.

  14. 14.

    Drozdzik M, Gröer C, Penski J, Lapczuk J, Ostrowski M, Lai Y, et al. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine. Mol Pharm. 2014;11(10):3547–55. https://doi.org/10.1021/mp500330y.

  15. 15.

    Brun V, Dupuis A, Adrait A, Marcellin M, Thomas D, Court M, et al. Isotope-labeled protein standards: toward absolute quantitative proteomics. Mol Cell Proteomics. 2007;6(12):2139–49. https://doi.org/10.1074/mcp.M700163-MCP200.

  16. 16.

    Ping L, Zhang H, Zhai L, Dammer EB, Duong DM, Li N, et al. Quantitative proteomics reveals significant changes in cell shape and an energy shift after IPTG induction via an optimized SILAC approach for Escherichia coli. J Proteome Res. 2013;12(12):5978–88. https://doi.org/10.1021/pr400775w.

  17. 17.

    Park SK, Liao L, Kim JY, Yates JR III. A computational approach to correct arginine-to-proline conversion in quantitative proteomics. Nat Methods. 2009;6(3):184. https://doi.org/10.1038/nmeth0309-184.

  18. 18.

    Mitchell CJ, Kim M-S, Na CH, Pandey A. PyQuant: a versatile framework for analysis of quantitative mass spectrometry data. Mol Cell Proteomics. 2016;15(8):2829–38. https://doi.org/10.1074/mcp.O115.056879.

  19. 19.

    Mousson F, Kolkman A, Pijnappel WP, Timmers HTM, Heck AJ. Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes. Mol Cell Proteomics. 2008;7(5):845–52. https://doi.org/10.1074/mcp.M700306-MCP200.

  20. 20.

    Gontan C, Mira-Bontenbal H, Magaraki A, Dupont C, Barakat TS, Rentmeester E, et al. REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice. Nat Commun. 2018;9(1):4752. https://doi.org/10.1038/s41467-018-07060-w.

  21. 21.

    Dannenmaier S, Stiller SB, Morgenstern M, Lübbert P, Oeljeklaus S, Wiedemann N, et al. Complete native stable isotope labeling by amino acids of Saccharomyces cerevisiae for global proteomic analysis. Anal Chem. 2018;90(17):10501–9. https://doi.org/10.1021/acs.analchem.8b02557.

  22. 22.

    Prokhorova TA, Rigbolt KT, Johansen PT, Henningsen J, Kratchmarova I, Kassem M, et al. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells. Mol Cell Proteomics. 2009;8(5):959–70. https://doi.org/10.1074/mcp.M800287-MCP200.

  23. 23.

    Piechura H, Oeljeklaus S, Warscheid B. SILAC for the study of mammalian cell lines and yeast protein complexes. In: Quantitative methods in proteomics. Springer; 2012. p. 201–21. https://doi.org/10.1007/978-1-61779-885-6_14.

  24. 24.

    Rutherfurd SM, Gilani GS. Amino acid analysis. Curr Protoc Protein Sci. 2009;58(1):11.19. 11–37. https://doi.org/10.1002/0471140864.ps1109s58.

  25. 25.

    Wolfe RR, Rutherfurd SM, Kim I-Y, Moughan PJ. Protein quality as determined by the digestible indispensable amino acid score: evaluation of factors underlying the calculation. Nutr Rev. 2016;74(9):584–99. https://doi.org/10.1093/nutrit/nuw022.

  26. 26.

    Calderón-Celis F, Encinar JR, Sanz-Medel A. Standardization approaches in absolute quantitative proteomics with mass spectrometry. Mass Spectrom Rev. 2018;37(6):715–37. https://doi.org/10.1002/mas.21542.

  27. 27.

    Overmyer KA, Tyanova S, Hebert AS, Westphall MS, Cox J, Coon JJ. Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice. Nat Protoc. 2018;13(2):293. https://doi.org/10.1038/nprot.2017.121.

  28. 28.

    Ibarrola N, Kalume DE, Gronborg M, Iwahori A, Pandey A. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture. Anal Chem. 2003;75(22):6043–9. https://doi.org/10.1021/ac034931f.

  29. 29.

    Burkitt WI, Pritchard C, Arsene C, Henrion A, Bunk D, O’Connor G. Toward Système International d’Unité-traceable protein quantification: from amino acids to proteins. Anal Biochem. 2008;376(2):242–51. https://doi.org/10.1016/j.ab.2008.02.010.

  30. 30.

    Holden MJ, Rabb SA, Tewari YB, Winchester MR. Traceable phosphorus measurements by ICP-OES and HPLC for the quantitation of DNA. Anal Chem. 2007;79(4):1536–41. https://doi.org/10.1021/ac061463b.

  31. 31.

    Liang Y, Yang L, Wang Q. An ongoing path of element-labeling/tagging strategies toward quantitative bioanalysis using ICP-MS. Appl Spectrosc Rev. 2016;51(2):117–28. https://doi.org/10.1080/05704928.2015.1105244.

  32. 32.

    Linscheid MW. Molecules and elements for quantitative bioanalysis: the allure of using electrospray, MALDI, and ICP mass spectrometry side-by-side. Mass Spectrom Rev. 2019;38(2):169–86. https://doi.org/10.1002/mas.21567.

  33. 33.

    Yan X, Luo Y, Zhang Z, Li Z, Luo Q, Yang L, et al. Europium-labeled activity-based probe through click chemistry: absolute serine protease quantification using 153Eu isotope dilution ICP/MS. Angew Chem Int Ed. 2012;51(14):3358–63. https://doi.org/10.1002/anie.201108277.

  34. 34.

    Zhang H, Gao N, Tian X, Liu T, Fang Y, Zhou J, et al. Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo. Sci Rep-UK. 2015;5:17671. https://doi.org/10.1038/srep17671.

  35. 35.

    Yan H, Hao F, Cao Q, Li J, Li N, Tian F, et al. A novel method for identification and relative quantification of N-terminal peptides using metal-element-chelated tags coupled with mass spectrometry. Sci China Chem. 2014;57(5):708–17. https://doi.org/10.1007/s11426-013-5049-8.

  36. 36.

    Yang L, Peng Y, Jiao J, Tao T, Yao J, Zhang Y, et al. Metallic element chelated tag labeling (MeCTL) for quantitation of N-glycans in MALDI-MS. Anal Chem. 2017;89(14):7470–6. https://doi.org/10.1021/acs.analchem.7b01051.

  37. 37.

    Wei J, Ding C, Zhang J, Mi W, Zhao Y, Liu M, et al. High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach. Anal Bioanal Chem. 2014;406(17):4183–93. https://doi.org/10.1007/s00216-014-7784-x.

  38. 38.

    Wang X, Wang X, Qin W, Lin H, Wang J, Wei J, et al. Metal-tag labeling coupled with multiple reaction monitoring-mass spectrometry for absolute quantitation of proteins. Analyst. 2013;138(18):5309–17. https://doi.org/10.1039/c3an00613a.

  39. 39.

    Brancia FL, Oliver SG, Gaskell SJ. Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides. Rapid Commun Mass Spectrom. 2000;14(21):2070–3. https://doi.org/10.1002/1097-0231(20001115)14:21<2070::AID-RCM133>3.0.CO;2-G.

  40. 40.

    Wang H, Zhang H, Li J, Wei J, Zhai R, Peng B, et al. A new calibration curve calculation method for absolute quantification of drug metabolizing enzymes in human liver microsomes by stable isotope dilution mass spectrometry. Anal Methods-UK. 2015;7(14):5934–41. https://doi.org/10.1039/C5AY00664C.

  41. 41.

    Rauniyar N, McClatchy DB, Yates JR III. Stable isotope labeling of mammals (SILAM) for in vivo quantitative proteomic analysis. Methods. 2013;61(3):260–8. https://doi.org/10.1016/j.ymeth.2013.03.008.

  42. 42.

    Choe LH, Aggarwal K, Franck Z, Lee KH. A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells. Electrophoresis. 2005;26(12):2437–49. https://doi.org/10.1002/elps.200410336.

  43. 43.

    Pieper S, Beck S, Ahrends R, Scheler C, Linscheid MW. Fragmentation behavior of metal-coded affinity tag (MeCAT)-labeled peptides. Rapid Commun Mass Spectrom. 2009;23(13):2045–52. https://doi.org/10.1002/rcm.4118.

  44. 44.

    Rivers J, Simpson DM, Robertson DH, Gaskell SJ, Beynon RJ. Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT. Mol Cell Proteomics. 2007;6(8):1416–27. https://doi.org/10.1074/mcp.M600456-MCP200.

  45. 45.

    Carroll KM, Simpson DM, Eyers CE, Knight CG, Brownridge P, Dunn WB, et al. Absolute quantification of the glycolytic pathway in yeast: deployment of a complete QconCAT approach. Mol Cell Proteomics. 2011;10(12):M111. 007633. https://doi.org/10.1074/mcp.M111.007633-1.

  46. 46.

    Ohtsuki S, Schaefer O, Kawakami H, Inoue T, Liehner S, Saito A, et al. 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. 2012;40(1):83–92. https://doi.org/10.1124/dmd.111.042259.

  47. 47.

    Kawakami H, Ohtsuki S, Kamiie J, Suzuki T, Abe T, Terasaki T. 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. 2011;100(1):341–52. https://doi.org/10.1002/jps.22255.

  48. 48.

    Achour B, Al Feteisi H, Lanucara F, Rostami-Hodjegan A, Barber J. Global proteomic analysis of human liver microsomes: rapid characterization and quantification of hepatic drug-metabolizing enzymes. Drug Metab Dispos. 2017;45(6):666–75. https://doi.org/10.1124/dmd.116.074732.

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This study was supported by the National Key R&D Program of China (No. 2017YFA0505002, 2018YFF0212505, 2018YFC0910302, 2016YFA0501403, and 2017YFC0906703), the National Key Laboratory of Proteomics Grant SKLP-K201706 and the National Natural Science Foundation of China (No. 21675172) and Innovation Project 16CXZ207. The authors would also like to thank the Shiyanjia Lab (www.shiyanjia.com) for the ICP-OES analysis.

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Correspondence to Weijie Qin.

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Lv, Y., Zhang, H., Wang, G. et al. A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferase enzymes in the human liver. Anal Bioanal Chem (2020). https://doi.org/10.1007/s00216-020-02445-7

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  • QconCAT
  • Absolute quantification
  • Lanthanide element labeling
  • Mass spectrometry
  • Drug-metabolizing enzymes