Multichannel Liquid Chromatography–Tandem Mass Spectrometry Cocktail Method for Comprehensive Substrate Characterization of Multidrug Resistance-Associated Protein 4 Transporter
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To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4).
Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography–tandem mass spectrometry.
From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4.
The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.
Key wordsATP-binding cassette transporter human multidrug resistance-associated protein 4 liquid chromatography–tandem mass spectrometry substrate screening vesicle uptake study
blood–cerebrospinal fluid barrier
central nervous system
liquid chromatography–mass spectrometry
liquid chromatography–tandem mass spectrometry
multiple reaction monitoring
multidrug resistance-associated protein
membrane uptake-index difference
organic anion transporter
We thank H. Yabuuchi (GenoMembrane, Yokohama, Japan) for kindly supplying the human MRP4-expressing membrane vesicles and the control vesicles. We also thank N. Funayama for secretarial assistance. This study was supported, in part, by a Grant-in-Aid for Scientific Research on Priority Area 17081002 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and by a 21st Century Center of Excellence (COE) Program Grant from the Japan Society for the Promotion of Science. This study was also supported, in part, by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.
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