We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/lg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/lg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/lg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.

A sensitive and simultaneous quantification method was developed for membrane proteins. By using this method, we constructed a quantitative atlas of membrane transporter proteins at the blood-brain barrier, liver and kidney in mouse. This technology is expected to have major implications for various fields of biomedical science.

We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

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Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx).

The purpose of this study was to determine absolute protein expression levels of transporters in rat choroid plexus, that is, the blood-cerebrospinal fluid barrier, and to compare them with the levels in the human choroid plexus. Plasma membrane fractions were prepared from pooled, freshly isolated choroid plexuses of 30 male Wistar rats and from frozen choroid plexus of one male human donor. Protein expression levels of 54 rat and 121 human molecules were measured, using a quantitative targeted absolute proteomics technique. In rat, oatp1a5 showed the most abundant protein expression (30.3 fmol/lg protein), and its expression level was 3.1-, 4.5-, 5.5-, 8.4-, 9.0-, 9.9-, 22-, 91-, and 95-fold greater than those of glut1, oatp1c1, mrp1, mct1, oat3, pept2, mrp4, bcrp, and mdr1a, respectively. OATP1A2 (a possible homolog of rat oatp1a5), OATP1C1 and PEPT2 were not detected in human choroid plexus. MRP1, OAT3, and MRP4 showed 4.0-, 1.8-, and 1.7-fold smaller expression levels in human than rat, respectively. MATE1 was detected in human, but not rat, and its expression level (8.61 fmol/lg protein) was the highest among the xenobiotic transporters examined in human choroid plexus. These findings should be useful for understanding rat blood-cerebrospinal fluid barrier function and its differences from that in human. Keywords: blood-cerebrospinal fluid barrier, human-rat difference, MATE1, oatp1a5, PEPT2, protein expression level. The transport capacities of membrane transporters at the BCSFB have been evaluated using specific substrates and/or inhibitors of target transporters by means of several techniques, such as intracerebroventricular administration and uptake experiments using isolated choroid plexus (Kuroda et al. 2005;Tachikawa et al. 2012). However, potential overlaps of substrate specificity among different transporters, including ones with unknown function, together with the lack of specific substrates and inhibitors, make these Received February 8, 2015; revised manuscript received April 9, 2015; accepted April 13, 2015. Address correspondence and reprint requests to Tetsuya Terasaki, Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: terasaki.tetsuya@m.tohoku. ac.jpAbbreviations used: ABC, ATP-binding cassette; BBB, blood-brain barrier; BCSFB, blood-cerebrospinal fluid barrier; CNS, central nervous system; CSF, cerebrospinal fluid; LC-MS/MS, liquid chromatographytandem mass spectrometry; LQ, limit of quantification; QTAP, quantitative targeted absolute proteomics; SLC, solute carrier; SRM/MRM, selected/ multiple reaction monitoring; ULQ, under limit of quantification.