Multichannel liquid chromatography-tandem mass spectrometry cocktail method for comprehensive substrate characterization of multidrug resistance-associated protein 4 transporter.

摘要: To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates multidrug resistance-associated protein 4 (MRP4/ABCC4). Human MRP4-expressing membrane vesicles were incubated with mixture of 50 compounds, including methotrexate, known MRP4 substrate. The amounts transported simultaneously determined by liquid chromatography–tandem mass spectrometry. From 49 12 identified as substrate candidates in first screening. second screening was performed involving uptake using single quadrupole multichannel mode, third individual compounds multiple reaction monitoring mode. As result, eight additionally identified. Subsequently, fourth step, osmotic pressure-dependent transport demonstrated 18 (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate alacepril) among 20 candidates, thereby confirming them substrates. By contrast, uptakes meloxicam nateglinide did not depend on osmolarity, indicating that these substrates, but bound to MRP4. ABC transporters allowed identification