Quantitative Targeted Absolute Proteomic Analysis of Transporters

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Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model Sumio Ohtsuki, Chiemi Ikeda, Yasuo Uchida, Yumi Sakamoto, Florence Miller, Fabienne Glacial, Xavier Decleves, Jean-Michel Scherrmann, PierreOlivier Couraud, Yoshiyuki Kubo, Masanori Tachikawa, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 08 Nov 2012 Downloaded from http://pubs.acs.org on November 18, 2012

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Molecular Pharmaceutics

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model

Sumio Ohtsuki1,2, Chiemi Ikeda1, Yasuo Uchida1, Yumi Sakamoto1, Florence Miller3,4,5, Fabienne Glacial3,4,5, Xavier Decleves6,7, Jean-Michel Scherrmann6,7, Pierre-Olivier Couraud3,4,5, Yoshiyuki Kubo1, Masanori Tachikawa1, Tetsuya Terasaki1*

1

Division of Membrane Transport and Drug Targeting, Graduate School of

Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2

Department of Pharmaceutical Microbiology, Faculty of Life Sciences,

Kumamoto University, Kumamoto, Japan 3

INSERM, U1016, Institut Cochin, Paris, France

4

Cnrs, UMR8104, Paris, France

5

Univ Paris Descartes, Paris, France

6

Neuropsychopharmacologie des addictions (CNRS UMR 8206), Université

Paris Descartes, Faculté de Pharmacie, Paris, France 7

INSERM U705, Neuropsychopharmacologie des addictions, Paris, France

1

ACS Paragon Plus Environment


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*Corresponding author: Professor 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. TEL: +81-22-795-6831; FAX: +81-22-795-6886 E-mail: [email protected]

Running Title: Membrane protein levels in hCMEC/D3 as BBB model

Table of Contents Graphic

2


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Abstract 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 non-brain 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. 3



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Keywords: human blood-brain barrier, transporter, protein quantification, quantitative targeted absolute proteomics, multidrug resistant protein 1, breast cancer resistant protein, ATP-binding cassette transporter, receptor, junction protein, claudin-5

Abbreviations: ATP-binding cassette (ABC) transporters blood-brain barrier (BBB) human umbilical vein endothelial cells (HUVECs) liquid chromatography-tandem mass spectrometer (LC-MS/MS) quantitative targeted absolute proteomics (QTAP) multiple/selective reaction monitoring (MRM/SRM) Solute Carrier (SLC) transporters Under the limit of quantification (ULQ)

Footnote: T.T. is a full professor of Tohoku University, and S.O. is a full professor of Kumamoto University, and both are also directors of Proteomedix Frontiers. This research was not supported by Proteomedix Frontiers and their positions at Proteomedix Frontiers do not present any financial conflicts.

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Introduction The blood-brain barrier (BBB) is localized at the interface between blood and cerebral tissue, and is formed by the endothelial cells of brain microvessels, which display a unique phenotype characterized by the presence of intercellular tight junctions and the polarized expression of numerous transport systems. ATP binding cassette (ABC) transporters, such as MDR1, BCRP and MRP4, limit entry of drugs to the brain across the BBB.1 Among solute carrier (SLC) transporters, OAT3, Oatp1a4/Oatp-2 and Oatp1C1/Oatp-14 were reported to be involved in permeability of anionic drugs, opioids and/or thyroid hormones across the BBB.2-4 The BBB also expresses several SLC transporters for nutrients; GLUT1 for glucose, MCT1 for lactate and ketone bodies, and LAT1 for amino acids and L-dopa. The expressions of these transporters change depending upon nutrient availability and disease status.5-7 Furthermore, the BBB possesses receptor-mediated transporters, including insulin receptor, transferrin receptor

and

LRP1,

which

also

play

important

roles

in

delivering

macromolecules into the brain.8, 9 Our recent report demonstrated that the BBB expresses NPR-C, which is involved in elimination of atrial natriuretic peptide from the brain.10 A recent study documented marked species differences in mRNA expression of transporters in the brain microvessels of various species, including mouse, rat, porcine, bovine and human.11 Our recent quantitative targeted absolute proteomics (QTAP) study confirmed species differences in the expression of transporters at the protein level in human and mouse brain microvessels.12-14 Functions

and

protein

expressions

of

OAT3,

5


Oatp1a4/Oatp-2

and


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Oatp1C1/Oatp-14 were detected in mouse and rat BBB, but the protein expression levels of their homologues were below the limit of quantification in isolated human microvessels.12,

14

These observations highlighted the

importance of using human in vitro BBB models for analysis of the molecular functions and regulation of human BBB. The human brain endothelial cell line hCMEC/D3 retains most of the morphological and functional characteristics of brain endothelial cells (i.e., expression of tight junction proteins and multiple active transporters and receptors), even in monoculture without glial cells, and is considered to be a unique in vitro model of the human BBB.15-18 It has been widely used by many laboratories to analyze the functions of the human BBB and their modulation by pharmacological agents, xenobiotics, pathogens and other stimuli.19-23 Since comprehensive measurement of transport activities at the in vivo human BBB would be difficult, e.g., requiring the use of multiple appropriately labeled selective substrates, we considered that quantitative analysis and comparison of the expression levels of key transporters and other molecules would be an efficient alternative approach to validation of hCMEC/D3 cells as a BBB model, at least in terms of potential activities. We initially reported that hCMEC/D3 cells express mRNAs of MDR1, MRP1, MRP5 and BCRP, as well as the corresponding proteins.18 Subsequently, Carl et al. performed a comprehensive quantitative PCR study of transporters in hCMEC/D3 cells, confirming these initial observations, and reporting higher expression of MDR1, MRP1 and MRP4 mRNAs as compared with MRP5 and BCRP mRNAs.15 Dauchy et al. reported a similar expression pattern of ATP-binding cassette (ABC) transporter mRNAs in 6


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freshly isolated human brain microvessels, although BCRP mRNA was more highly expressed than MDR1, MRP1, MRP4 and MRP5 mRNAs.16 These reports suggest that the pattern of expression of ABC transporter mRNAs in hCMEC/D3 cells is at least qualitatively similar to that of human brain microvessels, although the expression levels differ. However, mRNA expression does not always correlate with protein expression and molecular function.24, 25 Because (i) membrane proteins, such as transporters, receptors and junction proteins, play important roles in BBB function and (ii) protein expression levels, rather than mRNA expression levels, are more closely related to protein function, we considered that quantitative analysis of key proteins in hCMEC/D3 cells and comparison of the results with those for freshly isolated human brain microvessels would be a rational strategy for further testing the validity of hCMEC/D3 cells as an in vitro human BBB model. For this purpose, we aimed to employ

our

spectrometer

recently

developed

liquid

(LC-MS/MS)-based

multiplexed-multiple/selective

chromatography-tandem

absolute

reaction

monitoring

quantification

mass by

(multiplexed-MRM/SRM)

method, which we have used to determine the protein expression levels of 114 membrane proteins in human brain microvessels.13, 14, 26 The purpose of the present study was, therefore, to comprehensively compare the protein expression levels of multiple key membrane proteins, including transporters, receptors and junction proteins, in hCMEC/D3 cells and human brain microvessels, as well as in human umbilical vein endothelial cells (HUVECs), as prototypical non-brain human endothelial cells.

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Materials and Methods Cell culture hCMEC/D3 cells were seeded at 1.5 x 106 cells on collagen type I coated 10 cm dishes. The cells were cultured in EBM-2 medium (Takara Bio, Shiga, Japan) supplemented with 2.5% fetal bovine serum, 0.025% VEGF, 0.025% R3-IGF, 0.025% hEGF, 0.01% hydrocortisone, 5 µg/mL bFGF, 10 mM HEPES and 1 % penicillin-streptomycin in an atmosphere of 95% air and 5% CO2 at 37°C for 3-4 days for routine culture. The culture period was extended to 6 days for cells used to prepare whole cell and plasma membrane fraction and the passage number was kept below 35.18 HUVECs (BD Biosciences, Bedford, MA) were seeded at 1.0-2.0 x 105 cells on 10 cm tissue culture dish (BD Biosciences), and cultured in HuMedia-EG2 supplemented with HuMedia-EG supplement set (Kurabo, Osaka, Japan) in an atmosphere of 95% air and 5% CO2 at 37°C for 4 days. The cells were collected for membrane preparation after 3 passages.

Preparation of membrane fraction Cells were scraped from culture dishes, and collected by centrifugation at 230 x g for 5 min at 4°C. The cells were suspended in suspension buffer (10 mM Tris-HCl (pH 7.4) containing 250 mM sucrose, 1 mM EGTA) with protease cocktail (SIGMA, St. Louis, MO), then lysed by nitrogen cavitation at 450 psi for 15 min at 4°C in a pressure vessel (Parr, Moline, IL). The homogenate was centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was ultracentrifuged at 100,000 × g for 40 min at 4°C. The resulting pellet was suspended in suspension buffer. The suspension was layered on top of 38% 8


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(w/v) sucrose solution and centrifuged at 100,000 × g for 40 min at 4°C. The turbid layer at the interface was recovered, suspended in suspension buffer, and centrifuged at 100,000 × g for 40 min at 4°C to obtain the plasma membrane fraction. Protein concentrations were measured by the Lowry method using the DC protein assay reagent (Bio-Rad, Hercules, CA).

LC-MS/MS-based protein quantification analysis. Protein quantitation of the target molecules was simultaneously performed by LC-MS/MS using our previously described multiplexed-MRM/SRM method.14 Protein expression levels were determined by quantifying specific target peptides produced by trypsin digestion. The absolute amount of each target peptide was determined by using an internal standard peptide, which is a stable isotope-labeled peptide with identical amino acid sequence to that of the corresponding target peptide. The target peptide for quantification was selected based on in-silico selection criteria as described previously.26 Quantification of human transporters and other membrane proteins was carried out under the MRM/SRM conditions reported previously.12, 14, 26 Protein samples (100 µg) were suspended in suspension buffer containing 7 M guanidium hydrochloride and 10 mM EDTA. Samples were reduced with dithiothreitol at room temperature for 60 min under a nitrogen atmosphere, and S-carbamoylmethylated with iodoacetamide at room temperature for 60 min. The alkylated proteins were precipitated with a mixture of methanol and chloroform. The precipitates were dissolved in 6 M urea, diluted with 100 mM Tris-HCl

(pH

8.0)

and

digested

with

tosylphenylalanyl

9


chloromethyl


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ketone-treated trypsin at an enzyme/substrate ratio of 1:100 at 37°C for 16 hours. The tryptic digests were spiked with stable isotope-labeled internal standard peptides and acidified with formic acid for analysis with the HPLC system, which was connected to an electrospray ionization triple quadrupole mass spectrometer (API5000, AB Sciex, Foster City, CA, USA) operated in positive ionization mode. LC was performed with C18 columns. Linear gradients of 1-50% acetonitrile in 0.1% formic acid were applied to elute the peptides at a flow rate of 50 µl/min. The mass spectrometer was set up to run a multiplexed-MRM/SRM experiment for peptides. The ion counts in the chromatograms were determined by using the quantitation procedures in Analyst software (AB Sciex). One specific peptide was selected for quantification of each target protein, and measured at 4 different MRM/SRM transitions. The amount of each peptide was determined as the average of 3 or 4 MRM/SRM transitions from one sample. In cases where signal peaks were obtained at only 2 or fewer transitions, the amount of peptide in the sample was defined as under the limit of quantification, unless otherwise noted in the tables. The value of the limit of quantification in each transition was defined as the value giving a peak area of 5000 counts for each protein sample, calculated by using the peak area of the isotope-labeled internal standard peptide in the corresponding transition of the same sample.

10


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Results Membrane protein expression levels in hCMEC/D3 cells The quantification of membrane proteins was initially examined using whole cell fraction of hCMEC/D3 cells (Table 1), since protein expression had been measured in whole cells of isolated human brain microvessels in our previous report.14 However, only 7 membrane proteins were found to be present at quantifiable levels. This result indicated that it would be necessary to prepare a plasma membrane fraction in order to quantify the expression levels of less abundant membrane proteins. The protein expression levels of 91 membrane proteins, i.e., 81 transporters, 7 receptors, 1 junction protein and 2 membrane marker proteins (Na+/K+ ATPase and γ-GTP) were measured in plasma membrane fractions of hCMEC/D3 cells. The quantification was performed by means of multiplexed MRM/SRM with the same specific peptides used in our previous report.14 As shown in Table 1, the protein expression levels of 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were determined, while the other membrane proteins (listed in Table 2) were below the limit of quantification. Among drug-related transporters, such as MDR1, MRPs and BCRP, MDR1 exhibited the highest protein expression in plasma membrane fraction of hCMEC/D3 cells. The expression levels of BCRP, MRP1 and MRP4 were 56.3%, 42.6% and 8.04% of that of MDR1. Among the ABC transporters, ABCA2 showed the highest expression. Among SLC transporters and receptors, GLUT1 and transferrin receptor, respectively, showed the most abundant expression.

11



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Comparison of membrane protein expression levels in hCMEC/D3 cells and isolated human brain microvessels. The protein expression levels of membrane proteins detected in hCMEC/D3 cells in the present study and those found previously in human brain microvessels14 are compared in Fig. 1. The protein expression levels in plasma membrane of hCMEC/D3 cells and in whole cell fractions of human brain microvessels were normalized by the expression level of Na+/K+ ATPase. Twelve membrane proteins were detected in both hCMEC/D3 cells and human brain microvessels. The normalized protein expression levels of these membrane proteins were within 4-fold difference, except for ENT1, transferrin receptor and claudin-5. The expression of claudin-5 was 5.27-fold greater in brain microvessels than in hCMEC/D3 cells. In contrast, the expression levels of ENT1 and transferrin receptor were 11.7 and 18.5-fold greater in hCMEC/D3 cells than in brain microvessels, respectively. ABCA3, ABCA6, MRP1 and ATA1 were detected only in hCMEC/D3 cells (Table 1, and Fig. 1), while ABCA8, LAT1, LRP1 and γ-GTP were detected only in human brain microvessels (Table 2, and Fig. 1).

Comparison of membrane protein expression levels in hCMEC/D3 cells and HUVECs. HUVECs are widely used as prototypical human vascular endothelial cells; here, they were used as non-brain cultured human endothelial cells for comparison with hCMEC/D3 cells. As shown in Table 3, the expression levels of 4 ABC transporters, 5 SLC transporters, 2 receptors, 1 junction protein and 1 12


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membrane marker were quantified in plasma membrane fraction of HUVECs. The protein expression levels normalized by that of Na+/K+ ATPase were compared in hCMEC/D3 cells and HUVECs (Fig. 2). Among 13 membrane proteins detected in HUVECs, the normalized protein expression levels of 12 were within 4-fold difference from those in hCMEC/D3 cells. In contrast, the protein expression level of GLUT1 was 15.2-fold higher in hCMEC/D3 cells than in HUVECs. Finally, MDR1 was detected in hCMEC/D3 cells, but was below the limit of quantification in HUVECs.

13



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Discussion The present study is the first to quantify the protein expression profile of transporters, receptors and junction proteins in cells of the immortalized human brain microvascular endothelial cell line hCMEC/D3 (Tables 1 and 2). Eleven transporters, receptors and junction proteins were detected in both hCMEC/D3 cells and brain microvessels, and the expression levels of 9 of them, including MDR1, BCRP and MRP4, were within 4-fold difference after normalization with respect to expression of the membrane marker protein Na+/K+ ATPase (Fig. 1). MDR1, BCRP and MRP4 play important roles in limiting drug permeability across the BBB,1 and a high activity of glucose transport by GLUT1 is recognized as a hallmark of the BBB.30 Indeed, hCMEC/D3 cells expressed these three ABC transporters and GLUT1 at high levels. In contrast, in peripheral microvascular endothelial cells, HUVECs, MDR1 was below the limit of quantification, as expected (Table 3 and Fig. 2), and GLUT1 was expressed at a

much

lower

level

than

in

hCMEC/D3

cells.

Furthermore,

OAT3,

Oatp1a4/Oatp-2 and Oatp1C1/Oatp-14 were reported to be involved in drug, opioid and/or thyroid hormone permeability across mouse and rat BBB, and the proteins were detected in mouse brain microvessels.2-4,

26

However, their

homologues were below the limit of quantification in isolated human microvessels,14 as is also the case in hCMEC/D3 cells. Overall, these results support the validity of hCMEC/D3 cells as a human BBB model for drug permeability across the human BBB. MRP1 was detected only in hCMEC/D3 cells, but not in human brain microvessels. It has been reported that MRP1 expression is induced during 14


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primary culture of microvascular endothelial cells.31 Therefore, MRP1 expression may have been induced in hCMEC/D3 cells during culture. As shown in Table 1, the protein level of MRP1 in plasma membrane fraction was greater than that of MRP4 and similar to that of BCRP, suggesting that the putative contribution of MRP1 activity in hCMEC/D3 cells should be considered in transport studies, depending on the compounds under investigation. Furthermore, in human brain microvessels, BCRP expression was higher by 1.34-fold than that of MDR1,14 whereas in plasma membrane fraction of hCMEC/D3 cells, MDR1 expression was higher by 1.77-fold than that of BCRP (Table 1). In conclusion, hCMEC/D3 cells retain expression of 3 major drug-related ABC transporters, MDR1, BCRP and MRP4, although the relative expression levels differ from those in human brain microvessels. These differences should also be taken into consideration in future studies on drug permeability using hCMEC/D3 cells. Carl et al. have reported the mRNA expression of multiple transporters in hCMEC/D3 cells.15 MDR1 exhibited the highest mRNA expression among ABC transporters, and this is consistent with our finding regarding expression of the corresponding protein in plasma membrane fraction (Table 1). However, mRNA expression of BCRP was lower than that of MRP1 and MRP4, confirming that mRNA expression levels do not always match corresponding protein expression levels. Furthermore, mRNA expression of MRP5 was reported in hCMEC/D3 cells, and its expression was higher than that of BCRP.15, 17,

18

However, we

found that MRP5 protein was below the limit of quantification in the plasma membrane fraction. Thus, our results further illustrate the importance of measuring protein expression levels for proper characterization of in vitro model 15



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Page 16 of 33

systems. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in hCMEC/D3 cells, although they were detected in human brain microvessels (Table 2 and Fig. 1), suggesting that expression of these proteins was suppressed in hCMEC/D3 cells. Since LAT1 mediates transport of CNS drugs, such as L-dopa,32 suppression of LAT1 expression could limit the usefulness of hCMEC/D3 cells for screening CNS drugs. As another possibility, it should be noted that the quantification limit of LAT1 normalized by Na+/K+ ATPase (0.828 / 31.4 = 0.0264) in the present analysis was close to, though greater than, the normalized expression level in isolated human microvessels (0.431 / 35.1 = 0.0123). Therefore, decreased sensitivity in LC-MS/MS analysis cannot be ruled out as an explanation of why LAT1 was below the quantification limit in hCMEC/D3 cells. The molecular mechanism of down-regulation in hCMEC/D3 cells is unknown. It has been reported that expression of LAT1 is induced by platelet-derived growth factor and is suppressed under arginine-deficient conditions.33,

34

Hypoxia also induced destabilization of LAT1 gene in brain capillary endothelial cells.35 Similarly, LRP1 expression on plasma membrane was reported to be induced by insulin treatment.28 Therefore, a variety of differences between in vitro and in vivo conditions, such as cytokines, hormones and amino acids, may be involved in down-regulation of these membrane molecules in the cells. Among amino acid transporters, LAT was below the quantification limit, while ATA1 was detected in hCMEC/D3 cells. But it remains possible that expression of amino acid transporters, including amino acid transporters not quantified in 16


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the present study, changes depending upon the culture conditions. Further comprehensive protein expression analysis of amino acid transporters in hCMEC/D3 cells under different culture conditions would be important for understanding the regulation of amino acid transport at the human BBB under various pathophysiological conditions. ABCA3 and ABCA6 were detected in hCMEC/D3 cells, while they were below the limit of quantification in isolated human brain microvessels. ABCA transporters are involved in lipid transport, and ABCA3 is regulated by glucocorticoid.36, 37 So, it is possible that ABCA transporters, such as ABCA2, 3, 6 and 8 are involved in lipid homeostasis in human brain capillary endothelial cells. These ABCA transporters are expressed not only in brain capillary endothelial cells, but also in brain parenchymal cells, such as astrocyte foot processes and pericytes.38 Contamination of the preparation of brain microvessels with brain parenchymal cells cannot be entirely excluded.4, 16 This is one of the limitations of using isolated human brain microvessels, and may account for some of the apparent differences in expression levels reported here between hCMEC/D3 cells and isolated brain microvessels. In the present study, we normalized the protein expression levels to that of Na+/K+ ATPase for comparison between cells and brain microvessels, on the assumption that the density of Na+/K+ ATPase on the plasma membrane was not different between the cells and brain microvessels. We used this approach because we could not obtain enough fresh human normal brain for preparation of plasma membrane fraction. We previously found very similar expression levels of Na+/K+ ATPase in whole cell fractions of isolated brain microvessels 17



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from human, monkey and mouse (36.1, 35.1 and 39.1 fmol/mg protein, respectively).14, 26, 39 The differences in expression of membrane proteins were mostly within 4-fold in the present study comparing hCMEC/D3 cells, HUVECs and brain microvessels. Without normalization to Na+/K+ ATPase, the maximum increase of expression level in plasma membrane fraction was 11.0-fold for ABCB1/MDR1 compared to whole cell fraction, while this increase was reduced to 2.56-fold after normalization, and the maximum difference in normalized expression levels between plasma membrane and whole cell fractions was 3.32-fold for SLC3A2/4F2hc. These findings support the rationale for normalization to Na+/K+ ATPase, although the validity of this procedure needs to be confirmed in further studies. The expression level of Na+/K+ ATPase in whole cell fraction was 4.8-fold less in hCMEC/D3 cells than in human brain microvessels. This is likely due to the greater amount of cytosolic protein in hCMEC/D3 cells, owing to differences in cell shape and volume between in vivo and in vitro. This would be consistent with the fact that most membrane proteins detected in human brain microvessels were below the quantification limit in whole cell fraction of hCMEC/D3 cells. However, down-regulation of Na+/K+ ATPase in hCMEC/D3 cells cannot be excluded. In conclusion, our results indicate that hCMEC/D3 cells retain protein expression of most transporters, receptors and junction proteins expressed in vivo at the human BBB, including MDR1, BCRP, MRP4, transferrin receptor, insulin receptor, GLUT1 and claudin-5. Therefore, our findings support the view that hCMEC/D3 cells are a useful model for examining mechanisms of drug transport across the human BBB, although there are some differences, such as 18


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increased expression of MRP1 and decreased expression of LRP-1 in these cells, compared with freshly isolated human brain microvessels. The biological function of proteins at the BBB is known to be regulated not only at the level of their expression, but also by post-translational modifications and regulation of subcellular localization. In future studies, we aim to apply MRM/SRM analysis to selectively quantify modified proteins, including phosphorylated proteins.40, The

present

results

also

demonstrate

that

our

41

LC-MS/MS-based

multiplexed-MRM/SRM method is suitable for absolute quantification of functionally important transporters, receptors and junction proteins in plasma membrane fraction of cultured cells. Since functional analysis of all transporters in human BBB in vivo is infeasible, quantitative analysis of functionally important proteins, e.g., transporters and tight junction proteins, of hCMEC/D3 and comparison of the results with those for brain microvessels represents an important contribution to validation of these cells as a human BBB model.

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Acknowledgements: This study was supported in part by Japan Society for the Promotion of Science (JSPS) and Centre National de la Recherche Scientifique (CNRS) under the Japan - France Basic Scientific Cooperation Program, and also by Grants-in-Aid for Scientific Research (A) [KAKENHI: 24249011] from JSPS; grants for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency (JST); the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.

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Table 1. Protein expression levels of membrane proteins in hCME/D3 cells Protein expression level (fmol/µg protein) Plasma membrane fr.

Whole cell fr

7.16 ± 0.63 b

ULQ (

Quantitative Targeted Absolute Proteomic Analysis of Transporters

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