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Research ArticleArticle

Label-Free but Still Constrained: Assessment of Global Proteomic Strategies for the Quantification of Hepatic Enzymes and Transporters

Jill Barber, Zubida M. Al-Majdoub, Narciso Couto, Areti-Maria Vasilogianni, Annika Tillmann, Sarah Alrubia, Amin Rostami-Hodjegan and Brahim Achour
Drug Metabolism and Disposition June 2022, 50 (6) 762-769; DOI: https://doi.org/10.1124/dmd.121.000780
Jill Barber
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Zubida M. Al-Majdoub
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Narciso Couto
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Areti-Maria Vasilogianni
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Annika Tillmann
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Sarah Alrubia
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Amin Rostami-Hodjegan
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Brahim Achour
Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester, Manchester, United Kingdom (J.B., Z.M.A.-M., N.C., A.-M.V., A.T., S.A., A.R.-H., B.A.) Simcyp Division, Certara, Sheffield, United Kingdom (A.R.-H.) and Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, the University of Rhode Island, Kingston, Rhode Island (B.A.)
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Abstract

Building and refining pharmacology models require “system” data derived from tissues and in vitro systems analyzed by quantitative proteomics. Label-free global proteomics offers a wide scope of analysis, allowing simultaneous quantification of thousands of proteins per sample. The data generated from such analysis offer comprehensive protein expression profiles that can address existing gaps in models. In this study, we assessed the performance of three widely used label-free proteomic methods, “high N” ion intensity approach (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA), in relation to the quantification of enzymes and transporters in 27 human liver microsomal samples. Global correlations between the three methods were highly significant (R2 > 0.70, P < 0.001, n = 2232 proteins). Absolute abundances of 57 pharmacokinetic targets measured by standard-based label-free methods (HiN and iBAQ) showed good agreement, whereas the TPA overestimated abundances by two- to threefold. Relative abundance distribution of enzymes was similar for the three methods, while differences were observed with TPA in the case of transporters. Variability (CV) was similar across methods, with consistent between-sample relative quantification. The back-calculated amount of protein in the samples based on each method was compared with the nominal protein amount analyzed in the proteomic workflow, revealing overall agreement with data from the HiN method with bovine serum albumin as standard. The findings herein present a critique of label-free proteomic data relevant to pharmacokinetics and evaluate the possibility of retrospective analysis of historic datasets.

SIGNIFICANCE STATEMENT This study provides useful insights for using label-free methods to generate abundance data applicable for populating pharmacokinetic models. The data demonstrated overall correlation between intensity-based label-free proteomic methods (HiN, iBAQ and TPA), whereas iBAQ and TPA overestimated the total amount of protein in the samples. The extent of overestimation can provide a means of normalization to support absolute quantification. Importantly, between-sample relative quantification was consistent (similar variability) across methods.

Footnotes

    • Received November 14, 2021.
    • Accepted March 4, 2022.
  • Supported by the Centre for Applied Pharmacokinetic Research (CAPKR) consortium (Z.M.A.-M., B.A.), Cancer Research UK (A.-M.V.), the Saudi Ministry of Education (S.A.), and DrugTrain funded by the European Union’s Horizon 2020 research and innovation program (A.T.).

  • dx.doi.org/10.1124/dmd.121.000780.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 50 (6)
Drug Metabolism and Disposition
Vol. 50, Issue 6
1 Jun 2022
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Research ArticleArticle

Critique of Label-Free Proteomics of Pharmacokinetic Targets

Jill Barber, Zubida M. Al-Majdoub, Narciso Couto, Areti-Maria Vasilogianni, Annika Tillmann, Sarah Alrubia, Amin Rostami-Hodjegan and Brahim Achour
Drug Metabolism and Disposition June 1, 2022, 50 (6) 762-769; DOI: https://doi.org/10.1124/dmd.121.000780

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Research ArticleArticle

Critique of Label-Free Proteomics of Pharmacokinetic Targets

Jill Barber, Zubida M. Al-Majdoub, Narciso Couto, Areti-Maria Vasilogianni, Annika Tillmann, Sarah Alrubia, Amin Rostami-Hodjegan and Brahim Achour
Drug Metabolism and Disposition June 1, 2022, 50 (6) 762-769; DOI: https://doi.org/10.1124/dmd.121.000780
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