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Research ArticlePerspective

Lost in Centrifugation: Accounting for Transporter Protein Losses in Quantitative Targeted Absolute Proteomics

Matthew D Harwood, Matthew R Russell, Sibylle Neuhoff, Geoffrey Warhurst and Amin Rostami-Hodjegan
Drug Metabolism and Disposition October 2014, 42 (10) 1766-1772; DOI: https://doi.org/10.1124/dmd.114.058446
Matthew D Harwood
Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom
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Matthew R Russell
Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom
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Sibylle Neuhoff
Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom
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Geoffrey Warhurst
Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom
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Amin Rostami-Hodjegan
Gut Barrier Group, Inflammation & Repair, University of Manchester, Salford Royal NHS Trust, Salford (M.D.H., G.W.), Simcyp Limited (a Certara Company), Blades Enterprise Centre, Sheffield, (M.D.H., S.N., A.R-H.), and Centre for Applied Pharmacokinetic Research, Manchester Pharmacy School, Manchester (M.R.R., A.R-H.), United Kingdom
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Abstract

In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay’s sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition.

Footnotes

    • Received April 4, 2014.
    • Accepted July 24, 2014.
  • This work was supported by a grant from The Royal Commission for the Exhibition of 1851.

  • This work was contributed to Orbito IMI project (http://www.imi.europa.eu/content/orbito) as a sideground.

  • dx.doi.org/10.1124/dmd.114.058446.

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 42 (10)
Drug Metabolism and Disposition
Vol. 42, Issue 10
1 Oct 2014
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Research ArticlePerspective

Accounting for Protein Loss in Absolute Abundance Analysis

Matthew D Harwood, Matthew R Russell, Sibylle Neuhoff, Geoffrey Warhurst and Amin Rostami-Hodjegan
Drug Metabolism and Disposition October 1, 2014, 42 (10) 1766-1772; DOI: https://doi.org/10.1124/dmd.114.058446

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Research ArticlePerspective

Accounting for Protein Loss in Absolute Abundance Analysis

Matthew D Harwood, Matthew R Russell, Sibylle Neuhoff, Geoffrey Warhurst and Amin Rostami-Hodjegan
Drug Metabolism and Disposition October 1, 2014, 42 (10) 1766-1772; DOI: https://doi.org/10.1124/dmd.114.058446
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    • Abstract
    • Introduction
    • Subcellular Fractionation in Membrane Proteomics
    • Protein Losses in Centrifugation: The Utility of Recovery Factors
    • Accounting for Protein Losses in QTAP Abundance Analysis
    • Correcting for Protein Losses in Centrifugation
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