MS Based Quantitation of ABC and SLC Transporter Proteins

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LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line Laura Pelkonen, Kazuki Sato, Mika Reinisalo, Heidi Kidron, Masanori Tachikawa, Michitoshi Watanabe, Yasuo Uchida, Arto Urtti, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00782 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Molecular Pharmaceutics

LC-MS/MS Based Quantitation of ABC and SLC transporter proteins in plasma membranes of cultured primary human retinal pigment epithelium cells and immortalized ARPE19 cell line AUTHOR NAMES Laura Pelkonen†£, Kazuki Sato§£, Mika Reinisalo†, Heidi Kidron‡, Masanori Tachikawa§, Michitoshi Watanabe§, Yasuo Uchida§, Arto Urtti†‡¤* and Tetsuya Terasaki§¤ AUTHOR ADDRESS †

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland

§

Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical

Sciences, Tohoku University, Sendai, Japan ‡

Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,

University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland *

E-mail: [email protected]

KEYWORDS Quantitative targeted absolute proteomics, retinal pigment epithelium, transporter, ocular pharmacokinetics, blood retina barrier

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ABSTRACT The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1 and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4 and Na+/K+ ATPase were up-regulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.


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INTRODUCTION Systemic drug delivery to the retina remains challenging due to the protective blood-retinal barrier (BRB) that includes two separate barriers, the inner and outer BRB. The inner BRB consists of the endothelium of the retinal vessels, whereas the outer BRB is formed by a polarized cell monolayer, the retinal pigment epithelium (RPE) that serves as a selective barrier between the choroidal blood stream and the photoreceptors. The tight junctions in the RPE restrict the paracellular diffusion of compounds from the blood stream to the retina, thereby protecting the photoreceptors from xenobiotics (1-3). The RPE transports metabolic products and water from the neural retina to blood and provides nutrients for the photoreceptors from the choroidal blood stream thereby maintaining ideal conditions for the photoreceptor cells (2). Due to its role in fluid, nutrient and metabolic waste transport system, many channels (e.g. aquaporin 1) and transporter proteins (e.g. GLUT1, MCT1, MCT3) are expressed in the RPE (3). Systemic drug delivery is rarely an option in the retinal drug treatment, because the BRB limits the access of drugs to the retina (4). Clinically, intravitreal injection is the most common method of drug delivery to the posterior eye segment. The RPE has an important role also in this case, since it restricts the access of drugs to the choroid and regulates drug elimination from the vitreous and retina to the blood stream. Active transport has been suggested to play role in the vitreal drug elimination (5) although a previous study suggested that active transport may have low significance in the clearance of intravitreal drugs (6). The RPE is also a potential target in the pharmaceutical treatments, e.g. in dry form of age-related macular degeneration (AMD) (7). ARPE19 is a widely used immortalized human RPE cell line. Even though this cell line is a well characterized RPE model (8), it is reported to down-regulate several RPE signature genes (9) and


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represents low TER (10) compared to primary RPE cells. Filter-cultured ARPE19 is somewhat leakier than bovine RPE-choroid, but the cell line is able to reproduce the effect of the molecular weight and lipophilicity on the permeability (12). Human fetal RPE (hfRPE) cells are considered as a superior RPE model (12), but there is no systematic data on drug permeability across hfRPE cell monolayers (11). Functionality of some efflux transporters has been shown in the ARPE19 and D407 cell lines (12-17) and in stem cell derived RPE model (13). Even though many reports describe the transporter mRNA and protein expression in RPE models (reviewed by (18)), the expression results vary among different RPE models, for example in the case of P-glycoprotein (P-gp, MDR1) (13, 14) (16, 17, 19, 20). Furthermore, the abundance and significance of the transporter proteins cannot be estimated based on mRNA levels or qualitative protein studies; quantification at protein level in plasma membranes of the RPE is needed. Quantitative targeted absolute proteomics (QTAP) provides means to determine absolute protein expression levels in biological samples (21). Recently, the technique was used to determine the transporter protein contents in the blood-brain barrier (22, 23), Caco-2 cells (24) and human hepatocytes (25). As the protein abundance may correlate with the transport activity (28), QTAP is a powerful tool in transporter field (26). Systematic quantitation of the transporter protein expression in the RPE cells has not been conducted earlier. The purpose of the present study was to quantify transporter protein expression in the plasma membranes of ARPE19 cell line and hfRPE cells using QTAP in order to evaluate the ARPE19 cell line as a RPE model for pharmaceutical studies.


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MATERIALS AND METHODS Materials. Standard unlabeled and stable isotope-labeled peptides for quantification in LCMS/MS were synthesized by Thermoelectron Corporation (Sedantrabe, Germany) with over 95% peptide purity. All other chemicals were commercial products of analytical grade. Cell culture. ARPE19 cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM-F12 (1:1) (Gibco BRL, Grand Island NY, USA) containing 10 % fetal bovine serum (Gibco BRL, Grand Island NY, USA), 2 mM l-glutamine (EuroClone, Pero, Italy), 100 U/ml penicillin and 100 U/ml streptomycin (EuroClone, Pero, Italy) on regular culture dishes. Passages 26-33 were used in the studies. Human fetal RPE (hfRPE) cells (HRPEpiC cells, lot 12612) were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in EpiCM medium (ScienCell Research Laboratories, San Diego, CA, USA). The cells were expanded for 10 days, and at passage 3, they were seeded on regular culture dishes at high seeding density (200 000 cells/cm2). Both cell lines were cultured for one month before experiments at 37°C in 5 % CO2 atmosphere. Culture areas of 530-660 cm2 and 5310 cm2 comprising of approximately 106-131 x 106 and 4.68 x 108 hfRPE and ARPE19 cells, respectively, were used in the studies. The hfRPE cells (p-3, one month in culture) were characterized in regard to differentiation and RPE-specific properties (for details, see the Supplementary Information). The hfRPE cells used in this study represent many important RPErelated properties, including pigmentation, photoreceptor outer segment (POS) phagocytosis, tight junction and microvilli formation and the expression RPE specific marker genes (RPE65, BEST1).


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Plasma membrane fraction preparation. Plasma membranes from ARPE19 and hfRPE cells were isolated with previously published method for human cerebral microvascular endothelial cell line (hCMEC/D3) (23) with minor modifications. Briefly, after one month in culture, the cells were washed twice with ice-cold PBS solution (Gibco BRL, Grand Island NY, USA) and removed from the culture plates with plastic cell scraper. The cells were pelleted with centrifugation (2500 g for 5 min at 4 °C) and introduced to hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl2) containing inhibitors (PMSF and protease inhibitor cocktail, Sigma Aldrich). The cells were lysed with nitrogen cavitation (Parr Instruments) at 450 psi with 15 min equilibration time. Cell debris, nuclei and mitochondria were removed with three consecutive centrifugations at 15 000 g 10 min at 4 °C (SorvallTM WX Ultra Centrifuge, T1250 Rotor, Thermo Fisher Scientific Inc., Waltham, MA USA). The supernatant was recovered and pelleted with ultracentrifugation at 100 000 g 40 min at 4 °C (SorvallTM WX Ultra Centrifuge, TH-641 Swinging Bucket Rotor, Thermo Fisher Scientific Inc., Waltham, MA USA). The pellet represented the crude membrane fraction. The crude membrane fraction was suspended into suspension buffer (10 mM Tris-HCl, 250 mM sucrose) using 25 G needle and layered on top of 38 % sucrose solution and centrifuged at 100 000 g 40 min at 4 °C (SorvallTM WX Ultra Centrifuge, TH-641 Swinging Bucket Rotor). The turbid layer was recovered, suspended into suspension buffer and pelleted by ultracentrifugation at 100 000 g 40 min at 4 °C (SorvallTM WX Ultra Centrifuge, TH-641 Swinging Bucket Rotor) in order to pellet the plasma membrane fraction. The protein concentrations were measured with Bradford or Lowry method (Bio-Rad Protein reagent, DC protein assay reagent, respectively, Bio-Rad, Hercules, CA, USA). Immunoblotting. Isolated fractions of ARPE19 cells were introduced to 1 % Triton-X-100 buffer containing inhibitors (PMSF and protease inhibitor cocktail, Sigma-Aldrich). Equal


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amounts of protein were loaded on the wells of SDS-PAGE gels (4-20 % Precise Tris-Glycine Gels, Thermo Fisher Scientific Inc. Waltham, MA, USA). Semi-dry electroblotting (Trans-Blot® SD Semi-Dry Transfer Cell, Bio-Rad) was used to transfer the proteins onto PVDF membrane (Bio-Rad). Primary antibody incubations were conducted at + 4 °C overnight (mouse-anti HSP60 1:1000, #ADI-SPA-806-F, Enzo Life Sciences Inc., Farmingdale, New York, USA; Anti-Na+/K+ ATPase α1, 1:1000, 05-369, Merck Millipore, Merck KGaA, Darmstadt, Germany; Calnexin Polyclonal Antibody 1:500, AB0041 and Cathepsin D Polyclonal Antibody 1:500, AB0043, SICGEN – Research and Development in Biotechnology Ltd, Cantanhede, Portugal). Secondary antibody incubations (goat anti-mouse, sc-2005; donkey anti-goat, sc-2020 and goat anti-rabbit sc-2030, 1:10 000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were conducted at room temperature for 45 min. AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE Healthcare) was used to detect the protein-antibody-complexes with chemiluminescence reaction (Image Quant RT ECL, GE Healthcare, Little Chalfont, UK). Protein quantification by SRM/MRM analysis with LC-MS/MS. The absolute expression values were analyzed by selective/multiple reaction monitoring (SRM/MRM) with the liquid chromatography - tandem mass spectrometry (LC-MS/MS) as previously described (27, 28). Plasma membrane fractions in hfRPE and ARPE19 were solubilized in 7 M guanidine hydrochloride, 0.5 M Tris-HCl (pH 8.5), 10 mM EDTA-Na, and the proteins were Scarbomoylmethylated with iodoacetamide following dithiothreitol as previously described (27). The alkylated proteins were mixed with methanol, chloroform and milli-Q water, and precipitated by centrifugation. The precipitates were dissolved with 6 M urea in 0.1 M Tris-HCl (pH 8.5), and diluted 5-fold with 0.1 M Tris-HCl (pH 8.5) containing Protease-Max surfactant (Promega, Madison, WI, USA; final concentration 0.05%). The dilutions were treated with lysyl


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endopeptidase (Lys-C: Wako Pure Chemical Industries, Osaka, Japan) at 30℃ for 3 h. The LysC digested peptides were treated with TPCK-treated trypsin (Promega, Madison, WI, USA) at 37℃ for 16 h. Tryptic digested peptides were mixed with internal standard peptides and trifluoroacetic acid (final concentration; 0.1%), and desalted with GL-Tip™ SDB and GL-Tip™ GC (GL Sciences Inc, Tokyo, Japan) according to the manufacturer’s instructions. The resultant desalted sample solvents were evaporated by centrifugal concentrator CC-105 (TOMY; Heat Low, 1 h) under vacuum, and dissolved with 0.1% formic acid/milli Q water for LC-MS/MS analysis. LC-MS/MS analysis was performed by coupling an Agilent1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) to a triple quadrupole mass spectrometer (QTRAP5500; AB SCIEX, Framingham, MA) equipped with Turbo V ion source (AB SCIEX). Peptide samples equivalent to 10-20 ug proteins were injected onto XBridge BEH300 C18 (1.0 mm i.d.×100 mm, 3.5µm particles, Waters) column and eluted with a linear gradients sequence. Mobile phase A and B respectively consisted of 0.1% formic acid in milli Q water and 0.1% formic acid in acetonitrile. The gradient sequence (130 min run time, at a flow rate of 50 µL/min) was as follow: (A:B), 99:1 for 5 min after injection, 40:60 at 65 min, 0:100 at 66 min and up to 68 min, 99:1 at 70min and up to 130 min. The eluted peptides were selectively and simultaneously analyzed by SRM/MRM mode with LC-MS/MS. One specific peptide for each target protein was chosen by applying our in silico selection criteria (21). These peptides were monitored with 4 different SRM/MRM transition sets (Q1/Q3-1, Q1/Q3-2, Q1/Q3-3, Q1/Q3-4: Table S1) derived from one set of stable isotopelabeled and unlabeled peptides. The dwell time of one transition was 10 ms. Chromatogram ion counts were determined by using an auto analysis system established in our laboratory (21).


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Signal peak with an over 5000 counts peak area, which was detected at the same retention time with stable isotope-labeled peptide, was defined as a positive peak. When positive peaks were observed in two to four sets of SRM/MRM transitions, the proteins were determined to be expressed in the samples. The protein expression levels were calculated as an average of quantitative values obtained from two to four SRM/MRM transitions in 3 independently prepared hfRPE samples and 5independently prepared ARPE19 samples. If the molecules were detected as positive in non or one SRM/MRM transition, the protein expressions were determined as under the limit of quantification (U.L.Q.) in LC-MS/MS, and the values were calculated as previously described (21, 28).

RESULTS Purity of the isolated membrane fractions. The plasma membrane isolation resulted in approximately 43-110 µg and 65-77 µg of proteins in the ARPE19 and hfRPE plasma membrane fractions, respectively. The immunoblots indicate that the membrane marker Na+/K+ ATPase had enriched in the plasma membrane fraction of ARPE19 cells whereas intracellular contamination from lysosomes (cathepsin D) or mitochondria (HSP60) was absent (Fig. 1). The endoplasmic reticulum marker (calnexin) was found in all isolated fractions (Fig. 1).


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Figure 1. Na+/K+ ATPase, calnexin, HSP60 and cathepsin D expression in the ARPE19 whole cell lysate and the isolated membrane fractions. The mitochondrial marker HSP60 is seen only in the whole cell lysate. In the plasma membrane fraction, also the lysosomal marker cathepsin D is absent, whereas a faint expression is seen in the crude membrane fraction. The ER marker calnexin and the plasma membrane marker Na+/K+ ATPase are seen in each fraction.

The purity of the plasma membrane fraction was confirmed with LC-MS/MS by studying the enrichment ratio in the plasma membrane fraction compared to the whole cell lysate in regard to the membrane marker Na+/K+ ATPase. The high enrichment ratio of Na+/K+ ATPase (average 4.77, calculated from Table S2) indicated that the plasma membrane fraction was concentrated with the plasma membrane proteins and the purification was successful. In addition, the enrichment ratio of Na+/K+ ATPase in the plasma membrane fraction was similar than reported previously with hCMEC/D3 cells (4.3-fold enrichment) (23). Furthermore, the number of detected transporter proteins was the highest in the plasma membrane fraction, thus the number of proteins detected increased along with the enrichment ratio indicating that the enrichment ratio was suitable to assess the purity of the isolated membrane fraction. The plasma membrane


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fraction/whole cell lysate enrichment ratio of Na+/K+ ATPase was not suitable for the purity assessment of the hfRPE cells since Na+/K+ ATPase is also expressed intracellularly in the RPE melanosomes (29), and the hfRPE cells represented pigmentation (for details, see the Supplementary Information regarding the hfRPE characterization). However, melanosomes are removed from the membrane fractions at the 15 000 g centrifugation, and therefore, the enrichment ratio between plasma membrane fraction and crude membrane fraction could be used in the evaluation of the plasma membrane fraction purity. The Na+/K+ ATPase expression was found to be 2.36 and 2.06-fold higher in the plasma membrane fractions compared to the crude membrane fractions in ARPE19 and hfRPE cells, respectively (Table S2). This indicated that the membrane proteins were similarly concentrated in the plasma membrane fractions of both cell lines. Transporter Expression Levels in hfRPE and ARPE19 cells. To investigate the transporter expression levels in the plasma membrane fractions of hfRPE and ARPE19 cells, these of 41 membrane proteins, including 15 ABC transporters and 24 SLC transporters, were measured by QTAP. Three and five independently isolated plasma membrane preparations of hfRPE and ARPE19 cells were analyzed, respectively (Tables 1 and 2). The protein expression levels of 16 membrane proteins were determined in hfRPE cells as shown in Table 1, and 25 membrane proteins were under the limit of quantification as shown in Table 3. Among transporters, MPR1, MRP4, MRP5, MRP7, OAT2 and MATE1 were detected in hfRPE cells, but P-gp, MDR3 and BCRP were not. MRP1 showed the highest expression levels followed by MATE1 and MRP5. Among transporters, glucose transporter (GLUT1), amino acid transporters (TAUT, CAT1 and LAT1), monocarboxylate transporters (MCT1 and MCT4),


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folate transporters (RFC1 and PCFT) were determined, and GLUT1 had the most abundant expression.

Table 1. Protein expression levels in the plasma membrane fractions of hfRPE Protein expression levels of plasma membrane fraction (fmol/µg protein) 1a ABCC1/MRP1 ABCC4/MRP4 ABCC5/MRP5 ABCC10/MRP7

1.78

2a

±

0.21

1.34

U.L.Q.(1.85

SLC19A1/RFC1

1.72

SLC22A7/OAT2

U.L.Q.(1.01 8.35

±

0.096

0.909

±

0.106

0.746

±

0.156

0.851

±

0.092

0.537

±

0.7

11.5

±

0.7

9.48

±

0.27

12.2

±

3.1

0.203

a

The protein expression levels (mean ± S.E.M) were calculated as the average of 3 or 4 MRM/SRM transitions. U.L.Q.: represents the values of the quantification limit (fmol/µg protein). b The average values (mean ± S.D.) were calculated as the average of hfRPE cells (n=3). c The expression levels were calculated as an average of 2 quantitative values obtained from two SRM/MRM transitions. d The expression levels were calculated from only a single SRM/MRM transition due to high noise levels at the other transitions. e The expression levels were calculated as the average values obtained from two samples.

On the other hand, the expression levels of 13 molecules were determined in the plasma membrane fractions of ARPE19 cells as shown in Table 2. Similar to the expression profile in hfRPE cells, MRP1 and GLUT1 showed the most abundant expression among drug transporters


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and nutrient transporters in ARPE19 cells. However, RFC1, MRP7 and OAT2 were not detected in ARPE19 cells. Table 2. Protein expression levels in the plasma membrane fractions of ARPE19 Protein expression levels of plasma membrane fraction (fmol/µg protein) 1a

2a

3a

4a

5a

Averageb

ABCC1/MRP1

5.20

±

0.92

1.92

±

0.17

2.91

±

0.29

1.83

±

0.13

8.12

±

0.97

4.00

±

ABCC4/MRP4

1.61

±

0.21

1.45

±

0.04

1.63

±

0.04

0.893

±

0.129

2.37

±

0.19

1.59

±

ABCC5/MRP5

2.68 0.53 d

0.836

±

0.157

1.37

±

0.08

U.L.Q.(

MS Based Quantitation of ABC and SLC Transporter Proteins

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