title: Quantitative targeted absolute proteomics for 28 human transporters in plasma membrane of Caco-2 cell monolayer cultured for 2, 3, and 4 weeks.
abstract: The purpose of the present study was to evaluate and compare the absolute protein expression levels of 28 drug-related transporters in Caco-2 cell monolayers cultured for 2, 3, and 4 weeks. Plasma membrane fractions of Caco-2 cells cultured on Transwell inserts for 2, 3 and 4 weeks were prepared and digested with trypsin, and then simultaneous absolute quantification of 28 transporters and Na(+)/K(+)-ATPase was conducted using our established quantitative targeted absolute proteomic technique. Nine transporters and Na(+)/K(+)-ATPase were detected. MDR1, BCRP, PEPT1, OSTα and OSTβ were highly expressed (greater than 1 fmol/μg protein), while MRP2, MRP4, OATP2B1 and MCT1 were moderately expressed (0.328-0.871 fmol/μg protein). No significant difference was observed in the protein expression levels of these transporters or Na(+)/K(+)-ATPase among the 2-, 3- and 4-week cultures. The other 19 transporters, including MRP1, MRP3, OATP1A2, OATP3A1, OATP4A1, and OATP1B3, were not detected. This information about the rank order of transporter protein expression will be useful to predict what transporter(s) are likely or unlikely to influence the permeability of various compounds across monolayers of Caco-2 cells, which are widely used in drug development studies.
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Electronic address: yuchida@m.tohoku.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.affiliation: Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Kamiie
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Ohmine
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Iwase
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010539:Permeability; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D026901:Membrane Transport Proteins
keywords: BCRP; Caco-2; Different culture period; LC-MS/MS; MDR1; OSTα/β; PEPT1; Protein expression level; QTAP; SRM/MRM analysis