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

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 wholecell 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.

Protein Abundance of Clinically Relevant Drug Transporters in the Human Liver and Intestine: A Comparative Analysis in Paired Tissue Specimens

“…Considering this information gap, we have recently published a study that characterized, for the first time, gene expression and protein abundance of clinically important transporters along the entire length of human intestine using paired tissues from organ donors (thereby avoiding the intersubject variability bias) . However, a considerable limitation of that analysis was protein determination in the isolated crude membrane fraction, which carried the risk of loss of protein and/or overestimation or underestimation of certain transporters compared to whole‐tissue lysates, as performed in this study, and has also been demonstrated in a recent comparative study on proteomics of absorption, distribution, metabolism, and excretion proteins . Therefore, we used the Filter‐Aided Sample Preparation (FASP) method to overcome this limitation in this study.…”

“…The resulting protein abundance data along with information on the yield of enriched membrane protein from a certain amount of hepatocytes or liver tissue could be translated into transporter abundance in hepatocytes or in the liver . However, there is a potential bias in these conversions/calculations . Therefore, the advantage of the current study is the availability of data from liver tissue lysates that could be directly used without further conversion.…”

Meta-Analysis of Expression of Hepatic Organic Anion–Transporting Polypeptide (OATP) Transporters in Cellular Systems Relative to Human Liver Tissue

“…However, a higher abundance should be expected, at least theoretically, in the plasma membrane fraction, which is anticipated to be more transporterenriched and functionally pertinent. Arguably, losses attributable to plasma membrane preparation methods could be responsible for this contradiction, as highlighted recently (Harwood et al, 2014). Plasma membrane may contain only a fraction of the total expressed transporter attributable to potential internalization reported for several (Bow et al, 2008;Kock et al, 2010;Choi et al, 2011).…”

In Vitro-In Vivo Extrapolation Scaling Factors for Intestinal P-Glycoprotein and Breast Cancer Resistance Protein: Part I: A Cross-Laboratory Comparison of Transporter-Protein Abundances and Relative Expression Factors in Human Intestine and Caco-2 Cells

“…To address whether there are laboratory-specific differences in generating system parameters for undertaking transporter-mediated IVIVE-PBPK, a multicenter study evaluating the consistency and comparability of the preparation steps and analytic outcome has been advocated (Harwood et al, 2014). However, to our knowledge, studies comparing the quantification of absolute transporter-protein abundances and the subsequent generation of REFs between laboratories for the same samples have not been reported in the literature yet.…”