title: Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.
abstract: To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs.
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Vineet
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Bhagwat
authors.initials: B
authors.lastname: Prasad
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Gabriela
authors.initials: G
authors.lastname: Patilea
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Anshul
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Laurent
authors.initials: L
authors.lastname: Salphati
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Raymond
authors.initials: R
authors.lastname: Evers
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.).
authors.forename: Cornelis E C A
authors.initials: CE
authors.lastname: Hop
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., B.P., G.P., J.D.U.); Drug Metabolism and Pharmacokinetics, Infection Innovative Medicines Unit, AstraZeneca Pharmaceuticals LLP, Waltham, Massachusetts (A.G.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (R.E.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, Californai (L.S., C.E.C.A.H.) jash@u.washington.edu.
authors.forename: Jashvant D
authors.initials: JD
authors.lastname: Unadkat
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D001692:Biological Transport; D016466:CHO Cells; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells; D003412:Cricetulus; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D004958:Estradiol / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D027381:Liver-Specific Organic Anion Transporter 1; D061985:Madin Darby Canine Kidney Cells; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D027361:Organic Anion Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001562:Benzimidazoles; D027381:Liver-Specific Organic Anion Transporter 1; D009363:Neoplasm Proteins; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; C503999:SLCO1B1 protein, human; D004958:Estradiol; C017807:bisbenzimide ethoxide trihydrochloride