title: Optimized approaches for quantification of drug transporters in tissues and cells by MRM proteomics.
abstract: Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.
authors.affiliation: Department of Pharmaceutics, University of Washington, P.O. Box 357610, Seattle, Washington, 98195, USA, bhagwat@uw.edu.
authors.forename: Bhagwat
authors.initials: B
authors.lastname: Prasad
authors.forename: Jashvant D
authors.initials: JD
authors.lastname: Unadkat
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002477:Cells / Q000737:chemistry; D006801:Humans; D026901:Membrane Transport Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D026901:Membrane Transport Proteins; D004364:Pharmaceutical Preparations
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