Abstract
The reliable quantification of drug transport proteins is a key element that is necessary to establish relative scaling factors for human pharmacokinetic predictions and to account for pharmacokinetic variations across populations. Herein, we report a detailed evaluation of targeted mass spectrometry-based quantification methods from the perspective of sample preparation for three major organic anion-transporting polypeptide (OATP) membrane proteins. We also demonstrate the first implementation of stable isotope labeling by amino acids in cell culture (SILAC) into the OATP quantification scheme, which hinges on label incorporation at the protein level to allow for a substantially accelerated sample preparation process with precise results. These methods will enable comparable quantifications to provide individual scaling factors as well as address potential inter-individual variability in OATP proteins that are central to transporter-mediated hepatic drug clearance. The evaluation procedures can also serve as a model for other proteolytically resistant transporters that have not been adequately detected by higher-throughput quantifications. In addition, results derived from various sample preparation conditions explicitly illustrate how the conditions have a differential impact on the quantification of different proteins. Consequently, the ratio between different proteins can be an artifact of the conditions rather than a reflection of the endogenous ratio for typical peptide-based quantifications where a stoichiometrical relationship between the proteolytic peptide and the protein is not established. To the extent that ratios determined under different sets of conditions can underlie different conclusions, our results highlight important limitations that have been underappreciated in recent drug transporter quantifications that extend peptide-based mass spectrometry tools beyond relative quantifications in order to directly compare the abundance of multiple drug transporters.
Highlights
The liver is a principal excretory organ that contributes to the elimination of endogenous substances, drugs, and metabolites through the processes of hepatic uptake, metabolism, and biliary excretion
Quantification of organic anion transporting polypeptide (OATP) proteins under various sample preparation conditions The time-course and denaturing conditions that initially consisted of heat denaturation in ammonium bicarbonate (ABC) buffer, 7 M Gdn, or 10% DOC, were examined with OATP1B1, OATP1B3, and OATP2B1
As determined from the final time point in our experiments, Gdn yielded quantification levels lower than controls while DOC provided the most efficient denaturation for OATP1B1 and OATP1B3 (Figure 2)
Summary
The liver is a principal excretory organ that contributes to the elimination of endogenous substances, drugs, and metabolites through the processes of hepatic uptake, metabolism, and biliary excretion. Given that in vitro model- and species-dependent variations in transporter expression constitute a significant hurdle for in vitro - in vivo extrapolations [5], the quantification of individual proteins becomes critical to establish scaling factors for pharmacokinetic predictions implemented during drug discovery as well as to account for pharmacokinetic variations across different populations. These scaling factors are important in biliary secretion predictions that are highly dependent upon in vitro and preclinical data due to lack of access to clinical bile samples. The general lack of selective compounds combined with the potential caveats of multiple binding underscores the need to pursue additional means of estimating contributions to total transport by using relative expression factors in a manner similar to relative activity factors [6]
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