Journal of Proteomics & Bioinformatics
Open Access

Abstract

Membrane Protein Quantification by Peptide-Based Mass Spectrometry Approaches: Studies on the Organic Anion-Transporting Polypeptide Family

Larissa M Balogh, Emi Kimoto, Jonathan Chupka, Hui Zhang and Yurong Lai

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.