Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes with Emphasis on Transporters

Physiology and Pharmacology Quantitative Protein Expression in the Human Retinal Pigment Epithelium: Comparison Between Apical and Basolateral Plasma Membranes With Emphasis on Transporters

Laura Hellinen,1 Kazuki Sato,2 Mika Reinisalo,1,3 Heidi Kidron,4 Kirsi Rilla,5 Masanori Tachikawa,2 Yasuo Uchida,2 Tetsuya Terasaki,2 and Arto Urtti1,4,6 1School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland 2Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 3Institute of Clinical Medicine, Department of Ophthalmology, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland 4Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland 5School of Medicine, Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland 6Laboratory of Biohybrid Technologies, Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russian Federation

Correspondence: Arto Urtti, Univer- PURPOSE. Retinal pigment epithelium (RPE) limits the xenobiotic entry from the systemic sity of Eastern Finland, Faculty of blood stream to the eye. RPE surface transporters can be important in ocular drug Health Sciences, School of Pharmacy, distribution, but it has been unclear whether they are expressed on the apical, basal, or both Yliopistonranta 1, P.O. Box 1627, cellular surfaces. In this paper, we provide quantitative comparison of apical and basolateral 70211 Kuopio, Finland; RPE surface proteomes. arto.urtti@uef.ﬁ. LH and KS contributed equally to the METHODS. We separated the apical and basolateral membranes of differentiated human fetal work presented here and should RPE (hfRPE) cells by combining apical membrane peeling and sucrose density gradient therefore be regarded as equivalent centrifugation. The membrane fractions were analyzed with quantitative targeted absolute authors. proteomics (QTAP) and sequential window acquisition of all theoretical fragment ion spectra Submitted: June 20, 2019 mass spectrometry (SWATH-MS) to reveal the membrane protein localization on the RPE cell Accepted: October 11, 2019 surfaces. We quantitated 15 transporters in unfractionated RPE cells and scaled their expression to tissue level. Citation: Hellinen L, Sato K, Reinisalo M, et al. Quantitative protein expres- RESULTS. Several proteins involved in visual cycle, cell adhesion, and ion and nutrient transport sion in the human retinal pigment were expressed on the hfRPE plasma membranes. Most drug transporters showed similar epithelium: comparison between api- abundance on both RPE surfaces, whereas large neutral amino acids transporter 1 (LAT1), p- cal and basolateral plasma membranes glycoprotein (P-gp), and monocarboxylate transporter 1 (MCT1) showed modest apical with emphasis on transporters. Invest enrichment. Many solute carriers (SLC) that are potential prodrug targets were present on Ophthalmol Vis Sci. 2019;60:5022– both cellular surfaces, whereas putative sodium-coupled neutral amino acid transporter 7 5034. https://doi.org/10.1167/ iovs.19-27328 (SNAT7) and riboﬂavin transporter (RFT3) were enriched on the basolateral and sodium- and chloride-dependent neutral and basic amino acid transporter (ATB0þ) on the apical membrane.

CONCLUSIONS. Comprehensive quantitative information of the RPE surface proteomes was reported for the first time. The scientific community can use the data to further increase understanding of the RPE functions. In addition, we provide insights for transporter protein localization in the human RPE and the significance for ocular pharmacokinetics. Keywords: blood–retinal barrier, retinal pigment epithelium, retinal cell culture, proteomics, transporter

etinal pigment epithelium (RPE) is a polarized cell of some transporter and channel proteins: the RPE provides R monolayer located between the photoreceptors of the nutrients for the photoreceptors from the choroidal blood flow retina and choroid. The apical microvilli of the RPE face the and removes metabolic waste products and excess water from photoreceptors whereas the basal surface faces Bruch’s the subretinal space.1,2 membrane on the choroidal side. The RPE is polarized; thus, The RPE is also an important tissue in ocular pharmacoki- many of its functions have specific direction either from the netics because it serves as the outer blood–retinal barrier. Tight apical-to-basolateral (from the retina into the choroidal blood junctions between the RPE cells limit the nonspecific diffusion flow) or basolateral-to-apical (from choroid into the retina) of molecules, thereby protecting the eye from xenobiotics direction. These include important vision supporting functions present in the systemic blood flow. As the RPE cells are

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damaged in ocular disorders, such as age-related macular TABLE 1. Antibodies Used in the Immunocytochemical Analysis degeneration (AMD) and diabetic retinopathy,3 the RPE itself is also an important drug target. It has been proposed that efflux Catalogue proteins on the RPE surface would serve as a functional Antibody Vendor Number Dilution component for the outer blood–retinal barrier by preventing 4 MCT1 Abcam ab90582 1:100 the ocular entry of their substrates. However, it is unclear LAT1 Cell Signaling Technology 5347S 1:50 which efflux transporters contribute and what is the extent of MDR1/P-gp Sigma Aldrich P7965-100UL 1:100 their impact. Many studies show conflicting results on the MRP1 Abcam ab3368 1:100 expression of the drug transporters, presumably due to the use 5 CD81 Abcam ab59477 1:100 of different cell models and antibody-based methods. In most MCT4 EMD Millipore AB3316P 1:100 cases, the localization of the transporters (apical or basal BEST1 Novus Biologicals NB300-164 1:50 surface) is unclear, and their transport directionality has not been investigated. In addition, the functional assays of efflux transport have been performed mostly in the RPE cell lines4,6 this study, and the schematic presentation is displayed in that may differ from the situation in vivo. Knowledge on Figure 1. The membrane separations were performed on two transporter localization on the RPE surface is important for understanding the ocular pharmacokinetics, because the separate assay days on which two individual membrane transporters may contribute to the inward (from the choroid separations were performed. This resulted in a total of four across the RPE into the eye) or outward (from the subretinal individually separated apical and basolateral plasma membrane space across the RPE into the choroid) transport depending on fractions and two whole cell lysates. The membrane fractions their localization. (n ¼ 4) were analyzed with both QTAP and SWATH, whereas Quantitative targeted absolute proteomics (QTAP) enables the whole cell lysates (n ¼ 2) were analyzed with QTAP. the quantitative assessment of protein expression. Thus, the The cells were rinsed twice with membrane-preserving expression of proteins can be compared between tissues and buffer (1 mM MgCl2 and 0.1 mM CaCl2 in PBS; Gibco BRL, cell models quantitatively, as in our previous study that Grand Island NY, USA). A nitrocellulose membrane (GE compared the transporter expression in the plasma mem- Healthcare, Chicago, IL, USA) was prewetted with sterile branes of the ARPE19 cell line and human fetal RPE (hfRPE).7 water and placed on top of the cell monolayer (Fig. 1A). However, our previous study did not provide information on Suction was used to remove excess water, and the cell plates transporter localization (apical or basolateral plasma mem- with nitrocellulose membranes were placed in the incubator brane), and the localization has remained mostly unclear in (þ378C, 5% CO2) for 5 minutes. After the incubation, the apical other earlier studies. In this paper, we quantified the membrane was peeled by lifting the nitrocellulose membrane expression of the previously studied 36 proteins in the apical from the cell monolayer (Fig. 1B). The peeling efficiency was and basolateral plasma membranes of primary RPE cells. These confirmed visually with light microscope. The apical mem- proteins include important drug transporting proteins (e.g., brane was scraped from the nitrocellulose membrane and multidrug resistance-associated proteins [MRPs], p-glycopro- collected with sterile water. The remaining cellular fraction tein [P-gp]) and other transporters that are important in RPE containing the basolateral membranes was rinsed twice with functions (e.g., GLUT1, MCTs). Because 15 of the detected PBS (Gibco BRL) and then scraped from the cell plates. The proteins were quantitated also in nonfractionated cell samples, membrane fractions were isolated with differential centrifuga- we scaled the expression of those 15 transporters to the RPE tion (Fig. 1C). The whole cells were removed with 1000g tissue level. In addition, we show the relative expression of centrifugation from both membrane fractions, and the >1300 proteins detected with SWATH-MS (sequential window supernatants were collected for further purification. The acquisition of all theoretical fragment ion spectra mass membrane fractions were purified by removing the light spectrometry) technology in the apical and basolateral plasma mitochondrial fraction with three consecutive 15,000g centri- membranes. SWATH-MS has recently been developed as a novel fugations for 10 minutes at þ48C, followed by membrane data-independent acquisition method and enables quantitative, pelleting at 100,000g for 40 minutes at þ48C (Sorvall WX Ultra 8–11 sensitive, and reproducible proteomic analysis. Centrifuge, T1250 Rotor; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The resulting membrane pellets contained purified apical plasma membrane fraction or crude basolateral MATERIALS AND METHODS membrane fraction (Fig. 1D). The crude basolateral membrane fraction was further purified with sucrose density gradient Cell Culture centrifugation as described earlier7 (Figs. 1E, 1F), resulting in Commercially available human fetal retinal pigment epithelial basolateral plasma membrane fraction. cells (hfRPE cells) were purchased from ScienCell (Carlsbad, The Bradford and Lowry method (Bio-Rad Protein reagent, CA, USA) (HRPEpiC, 6540) and expanded and maintained as DC protein assay reagent, respectively; Bio-Rad, Hercules, CA, described previously (in EpiCM medium 4101, at þ378Cin5% USA) was used to measure the protein concentrations. 7 CO2 atmosphere; ScienCell). At passage 3, the cells were 2 seeded at high density (285,000 cells/cm ) and retained in Immunofluorescence Analysis culture until fluid-filled domes appeared, indicating proper apical and basolateral polarity.12 This took 11 to 13 days after Cells were seeded on Ibidi l-slides at 200,000 cells/cm2 high-density seeding. (80826; Ibidi GmbH, Martinsried, Germany) and fixed with methanol after 2 weeks in culture. The immunofluorescence 7 Separation of Apical and Basolateral Plasma staining was conducted as described previously, with the Membrane Fractions primary antibodies described in Table 1 and specific Alexa Fluor (Thermo Fisher Scientific, Inc., Bleiswijk, The Nether- Apical and basolateral membrane fractions were separated lands) secondary antibodies. The cells were visualized with a from polarized hfRPE cell monolayers with a peeling method confocal microscope (Zeiss LSM 800; Carl Zeiss Microimaging described by Fong-ngern et al.13 The method was modified for GmbH, Jena, Germany).

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FIGURE 1. Schematic presentation of the apical and basolateral plasma membrane fraction separation. The RPE cells formed a monolayer in the culture. Prewetted nitrocellulose membrane was applied on top of the cells (A). The membrane was lifted resulting in the peeling of the apical microvilli (B). Both apical and basolateral fractions were purified with differential centrifugation (C), resulting in apical plasma membrane fraction and crude basolateral plasma membrane fractions (D). The crude basolateral fraction was further purified with sucrose density gradient centrifugation (E), resulting in the purified basolateral plasma membrane fraction (F).

Protein Quantification by QTAP Supplementary Table S3) to scale the protein expression onto human RPE tissue level (Table 2). The calculations are based on The protein digestion was performed as described previous- 14 our hfRPE cell culture areas and their corresponding total ly. A description of the procedures is detailed in the protein content (see Supplementary Table S4 for details) and Supplementary Methods. Proteins were quantitated with QTAP previously reported human RPE surface area15 (Table 2). as described previously.7,14 A description of the procedures is detailed in the Supplementary Methods. Transporters Displayed Nonpolarized Expression Comprehensive Quantitative Protein Expression in the RPE Profiling by SWATH-MS The isolated apical and basolateral plasma membrane fractions Procedures for comprehensive quantitative protein expression were analyzed separately by QTAP to determine whether their profiling by SWATH-MS are described in the Supplementary transporter abundances are similar. Among 36 analyzed Methods. proteins, 4 ABC transporters, 12 solute carriers (SLC) transporters, and Naþ/Kþ ATPase were quantified in both apical and basolateral membrane fractions (Table 2). The RESULTS comparison revealed that MRP1, MRP4, MRP5, GLUT1, 4F2hc, TAUT, CAT1, MCT4, MCT3, RFC1, OAT3, PCFT, MATE1, and hfRPE Cells Display Similar Transporter Protein Naþ/Kþ ATPase had similar abundance within 1.5-fold differ- Expression Levels After 2- and 4-Week Cultures ence in both membrane fractions (Fig. 2B; Table 2). However, monocarboxylate transporter 1 (MCT1), large neutral amino We compared the abundances of transporter proteins of the acids transporter 1 (LAT1), and MDR1 (P-gp) were more than cells that were cultured for 11 to 13 days (raw data values 1.5-fold enriched in the apical membrane compared with the presented in Supplementary Table S3 and mean values in Table basolateral membrane (Fig. 2B; Table 2). The expression levels 2) to the cells cultured for 28 to 31 days (reported values7)to of 11 ABC transporters, 7 SLC transporters, and membrane ensure that our cell model had appropriate transporter protein protein villin-1 remained under the limit of quantification expression profile after 2-week culture. The comparison was (ULQ; Supplementary Material; Fig. 2B). made between the whole cell lysate samples for the proteins whose expression levels were quantitated in both studies by QTAP (Fig. 2A). The transporter expression was stable between Marker Protein Localization in Intact Cells the different culture times (Fig. 2A), as the protein expression Confirms the Success of the Membrane Separation differences of each transporter were statistically insignificant (unpaired t-test with Welch’s correction; GraphPad Prism 5 We analyzed marker protein expression in intact cells cultured Software, San Diego, CA, USA). Two-week culture time was for 2 weeks after high-density seeding (Fig. 3) to verify the chosen because it resulted in proper differentiation of the cells success of plasma membrane separation. As in the QTAP without large fluid-filled domes that would have caused analyses, MCT1, LAT1, and P-gp displayed modest apical problems in membrane separation. enrichment, whereas MRP1 expression was detected mainly on the lateral cell surfaces, and MCT4 was present on all Expression of 15 Transporters Was Scaled Onto cellular surfaces. CD81, which was basally enriched in SWATH RPE Tissue Level analysis (Table 3), localized mainly on the basal cell membrane. RPE-specific basolateral marker protein bestrophin 1 (BEST1) We used the transporter expression levels in the whole cell was enriched to the basal side, indicating proper differentia- lysates determined with QTAP (Table 2, detailed data in tion (Fig. 3).

Downloaded from iovs.arvojournals.org on 09/27/2021 Downloaded fromiovs.arvojournals.org on09/27/2021 rnpre rti oaiaini h RPE the in Localization Protein Transporter TABLE 2. Transporter Protein Expression Levels in hfRPE Cells

Expression in the RPE, Scaling to the Tissue Level, Mean Expression in the Plasma Membrane Fractions, Assessment of the Localization

Abundance in Expression Expression Basolateral Apical Plasma Expression the Whole in the in the Plasma Membrane Membrane Ratio, Cell Lysate RPE fmol RPE fmol Fraction Fraction (fmol/lg Apical/ Protein/Gene (fmol/lg Expression, Expression, (Based on (Based on RPE (fmol/lg Protein),* Protein),* Basolateral, Abbreviation Protein)* fmol/Cell† fmol/cm2‡ RPE Area)§ Cell Count)† Mean 6 SEM Mean 6 SEM P Mean Localizationjj

ABCB1/MDR1/P-gp 0.145¶ 5.1 3 105 15 175 181 0.201 6 0.029¶ 0.392 6 0.051¶ 0.0172 1.95 Apical enrichment ABCC1/MRP1 1.25¶ 4.4 3 104 125 1,510 1,565 2.41 6 0.16 2.82 6 0.31 0.281 1.17 Equal abundance ABCC4/MRP4 0.129¶ 4.5 3 105 13 156 161 0.205 6 0.013 0.197 6 0.01 0.645 0.961 Equal abundance ABCC5/MRP5 0.447¶ 1.6 3 104 45 540 560 0.586 6 0.038 0.827 6 0.106 0.0771 1.41 Equal abundance SLC2A1/GLUT1 335¶ 0.12 33,604 404,594 419,320 981 6 60¶ 1141 6 84¶ 0.173 1.16 Equal abundance SLC3A2/4F2hc 4.12 1.5 3 103 413 4,976 5,157 10.9 6 1.7 14 6 1.6 0.237 1.28 Equal abundance SLC6A6/TAUT 0.282 9.9 3 105 28 341 353 0.298 6 0.071 0.238 6 0.053 0.523 0.799 Equal abundance SLC7A1/CAT1 1.46¶ 5.1 3 104 146 1,763 1,827 3.4 6 0.21 3.59 6 0.59 0.770 1.06 Equal abundance SLC7A5/LAT1 (ULQ < 0.936) NA (<3.3 3 104 )NA(<94) NA (<1130) NA (<1172) 1.09 6 0.17¶ 1.87 6 0.33# 0.0815 1.72 Apical enrichment SLC16A1/MCT1 3.83 1.3 3 103 384 4,626 4,794 9.96 6 0.66 16 6 2.06 0.0322 1.61 Apical enrichment SLC16A3/MCT4 10.1 3.6 3 103 1,013 12,198 12,642 17 6 3.9 17.2 6 2.6 0.969 1.01 Equal abundance SLC16A8/MCT3 0.166¶ 5.8 3 105 17 200 208 0.302 6 0.031¶ 0.269 6 0.052¶ 0.615 0.893 Equal abundance SLC19A1/RFC1 0.497 1.7 3 104 50 600 622 1.02 6 0.07 1.39 6 0.17 0.0929 1.36 Equal abundance SLC22A8/OAT3 (ULQ < 0.114) NA (<4.0 3 105 )NA(<11) NA (<138) NA (<143) 0.239 6 0.027¶ 0.25 6 0.063¶ 0.877 1.05 Equal abundance SLC46A1/PCFT 0.477¶ 1.7 3 104 48 576 597 1.89 6 0.09 1.74 6 0.27 0.631 0.924 Equal abundance SLC47A1/MATE1 0.268¶ 9.4 3 105 27 324 335 0.963 6 0.082¶ 1.17 6 0.05 0.0752 1.22 Equal abundance Naþ/Kþ ATPase 8.32 2.9 3 103 835 10,048 10,414 20.1 6 1.1 28.1 6 3.8 0.0895 1.4 Equal abundance NA, not applicable. * Whole cell lysate n ¼ 2 (raw data presented in Supplementary Material); apical and basolateral membrane fractions, n ¼ 3 to 4. The values display mean 6 SEM. † Expression calculated with the seeding density 285,000 cells/cm2 (assumption that the cells are not dividing after the high-density seeding). Total number of RPE cells in the adult human eyes was reported by Panda-Jonas et al.16: 3,556,290 6 490,700 cells (mean 6 SD; range, 2,130,500–4,653,200), which was used to evaluate the expression at tissue level. z The cell culture surface area and its total protein yield (55 cm2 corresponding to 5517 6 112 lg of total protein; see Supplementary Material for details). § RPE surface area was reported earlier in adult human eyes (1204 mm2)15. Our calculation is based on the surface area of the cell plate and the reported human RPE area.

jj Evaluation based on apical/basolateral expression ratio: more than 1.5-fold differences were considered as enrichment. Student’s t-test (2-tailed) was used to generate the P values evaluating the IOVS difference in expression levels between apical and basolateral membranes were analyzed unpaired.

¶ Some values that were located outside the standard curves were used for the calculation of the expression values. j eebr2019 December j o.60 Vol. j o 15 No. j 5025 Transporter Protein Localization in the RPE IOVS j December 2019 j Vol. 60 j No. 15 j 5026

FIGURE 2. (A) Transporter protein expression is similar in the RPE cells after 2 and 4 weeks in culture. Transporter protein expression in hfRPE cells cultured for 2 weeks (11–13 days, two biological replicates) and 4 weeks (28–31 days, three biological replicates) were compared after high- density seeding at passage 3. The protein abundances were determined with QTAP (cells cultured for 2 weeks; Table 2) and the abundances of the cells cultured for 4 weeks were reported earlier.7 The differences of each transporter protein abundance between 2- and 4-week culture times were statistically insigniﬁcant (unpaired t-test with Welch’s correction; GraphPad Prism 5 Software). (B) Most transporter proteins displayed nonpolarized expression in the RPE. Protein expression levels (data extracted from Table 2) between the apical and basolateral membrane fractions of hfRPE cells

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were compared. Each value is described as mean 6 SEM (n ¼ 3-4). The solid line represents the equal expression levels, and the broken lines represent 1.5-fold differences. More than 1.5-fold differences are described with black circles, whereas smaller differences are represented with gray circles. The expression level of GLUT1 was detected by QTAP, but it is not shown in this figure. (C, D) Protein expression ratios (apical/ basolateral) detected with SWATH-MS. Dotted lines indicate 1.5-fold and solid lines indicate 3-fold expression differences, respectively. The apical/ basolateral expression ratio of all the detected 1201 proteins were classified into transporters, receptors, other transmembrane proteins, and nontransmembrane proteins (C). (D) Selected important RPE proteins were classified according to their function (expression ratios of each protein presented in Table 3).

SWATH-MS Revealed the Expression and DISCUSSION Localization of Important RPE Proteins and In this study, we present quantitative differences in the hfRPE Transporters Relevant for Ocular Drug Delivery apical and basolateral plasma membrane proteomes deter- To make a comprehensive protein expression atlas and mined with SWATH-MS. In total, 1201 proteins were identified determine the protein localization, the apical and basolateral in both membrane fractions, providing important database for membrane fractions of hfRPE cells were analyzed with SWATH- understanding the cellular functions of the RPE. Furthermore, MS. We identified 1201 proteins in both apical and basolateral our SWATH data can be further used to map different functions membranes of hfRPE cells and determined their relative on the cellular membranes via pathway analysis. We also used expression differences. The validation of SWATH-MS results QTAP to determine transporter protein abundances separately was conducted by comparing the transporter expression ratios on both cellular surfaces and showed that many important obtained by SWATH-MS with those determined by QTAP (for drug transporting membrane proteins are present on both details, see Supplementary Material). sides of the hfRPE cell monolayer. The finding is important for All the proteins detected with SWATH-MS and their ocular pharmacokinetics as the RPE forms the outer blood– expression ratios (apical/basolateral) are presented in Supple- retinal barrier and drug permeation across the RPE is an mentary Tables S4 to S7. In total, 29 SLC and 3 ABC important factor in the ocular drug distribution. transporters and 55 receptors were identified. We detected additional 251 transmembrane and 863 nontransmembrane Membrane Separation and Polarization of the Cell proteins in the hfRPE apical and basolateral membranes and Model calculated their relative expression differences (Supplementary Tables S6, S7). Graphical presentation of the apical and We performed the hfRPE membrane separation once the cell basolateral expression ratios of detected transporters, recep- cultures had differentiated properly: fractionation was con- tors, other transmembrane proteins, and nontransmembrane ducted when the first signs of dome formation were evident in proteins is illustrated in Figure 2C. We selected important RPE the cultures at 11 to 13 days after high-density seeding. This proteins and classified them according to their role into six assured proper polarity of the cells and successful isolation. In different categories (perception of light stimulus, extracellular line with earlier literature on hfRPE cell differentiation in 11 to matrix and adhesion, pigmentation, phagocytosis, transporters 14 days,12 we detected many proteins that are important for and ion channels, and metal ion homeostasis; Fig. 2D; Table 3). RPE function (Table 3), indicating proper cellular maturity. The The classification was based on the categorization presented cells displayed apical enrichment of MCT1 and basal enrich- earlier by Hongisto et al.17 The relative expression of apical and ment BEST1 (Fig. 3), both considered to be RPE polarization basolateral membrane transporters that have clinical drugs as markers.33 In addition, the total transporter protein content in their substrates or are potential prodrug targets are presented the cells did not differ significantly between 2- and 4-week in Table 4. culture times (Fig. 2A). Taken together, the culture time, dome

FIGURE 3. Localization of marker proteins in hfRPE cells confirmed membrane separation success and proper cell differentiation. Immunofluorescence analysis showed apical enrichment of LAT1 (top two image panels), P-gp (third row), and MCT1 (bottom panel). MRP1 was found mostly on the lateral cell surfaces (third image panel). CD81 (first row) was enriched in the basal plasma membrane, whereas MCT4 (bottom panel) was detected on all cell surfaces (not included in the overview image on the right). Scale bars denote 20 lm.

Downloaded from iovs.arvojournals.org on 09/27/2021 Downloaded fromiovs.arvojournals.org on09/27/2021 rnpre rti oaiaini h RPE the in Localization Protein Transporter TABLE 3. Localization of Selected Proteins Involved in Important RPE Functions or Involved in Macular Degeneration Detected With SWATH-MS

Uniprot Expression Ratio Total Detected Accession Protein/Gene (Apical/Baso Transition Peptide RPE Function/Role No. Abbreviation Protein Name lateral) 6 SEM† Number‡ Number§ P Localizationjj in the RPE Condition¶

Selected important RPE proteins# Q07954 LRP1 Prolow-density lipoprotein 1.24 6 0.02 139 31 7.74E-29 Equal abundance Perception of light stimulus receptor-related protein 1 Q8TC12 RDH11 Retinol dehydrogenase 11 1.50 6 0.01 4 1 2.3E-10 Modest apical enrichment Perception of light stimulus Q8NBN7 RDH13 Retinol dehydrogenase 13 1.46 6 0.01 3 1 0.000000471 Equal abundance Perception of light stimulus Q9HBH5 RDH14 Retinol dehydrogenase 14 0.973 6 0.025 10 2 0.124 Equal abundance Perception of light stimulus P05362 ICAM1 Intercellular adhesion 1.29 6 0.06 43 9 0.00000908 Equal abundance ECM and adhesion molecule 1 P06756 ITGAV* Integrin alpha-V 1.16 6 0.04 66 14 0.000202 Equal abundance ECM and adhesion, phagocytosis P26012 ITGB8 Integrin beta-8 1.15 6 0.05 6 1 0.00145 Equal abundance ECM and adhesion P05556 ITGB1 Integrin beta-1 0.704 6 0.031 55 11 9.58E-22 Equal abundance ECM and adhesion P09382 LGALS1 Galectin-1 0.938 6 0.054 8 2 0.0767 Equal abundance ECM and adhesion P17643 TYRP1* 5,6-dihydroxyindole-2- 0.722 6 0.013 13 3 3.07E-19 Equal abundance Pigmentation carboxylic acid oxidase; DHICA oxidase, tyrosinase-related protein 1 P40967 PMEL* Melanocyte protein 4.20 6 0.45 17 4 6.86E-13 Mainly apical Pigmentation P14679 TYR Tyrosinase 0.568 6 0.023 34 8 1.35E-27 Modest basal enrichment Pigmentation P51810 GPR143* G-protein coupled receptor 0.543 6 0.040 25 5 5.35E-15 Modest basal enrichment Pigmentation 143 P60033 CD81 CD81 antigen 0.223 6 0.017 4 1 1.96E-08 Mainly basal Phagocytosis Q14108 SCARB2 Lysosome membrane 0.809 6 0.073 30 6 0.00000899 Equal abundance Phagocytosis protein 2 P13473 LAMP2* Lysosome-associated 0.600 6 0.033 23 4 8.95E-19 Modest basal enrichment Phagocytosis membrane glycoprotein 2 Q08431 MFGE8 Lactadherin 0.412 6 0.030 26 6 1.85E-25 Basal enrichment Phagocytosis IOVS P15311 EZR Ezrin 1.22 6 0.02 55 12 3.98E-20 Equal abundance Phagocytosis j

P16070 CD44 CD44 antigen 1.14 6 0.14 18 4 0.769 Equal abundance Phagocytosis 2019 December P07339 CTSD Cathepsin D 1.87 6 0.04 10 2 3.58E-20 Modest apical enrichment Phagocytosis P05023 ATP1A1 Sodium/potassium- 1.13 6 0.01 29 6 1.32E-22 Equal abundance Transporters and ion transporting ATPase channels subunit alpha-1 P05026 ATP1B1 Sodium/potassium- 1.20 6 0.02 30 6 1.58E-28 Equal abundance Transporters and ion

transporting ATPase channels j subunit beta-1 60 Vol. O60928 KCNJ13, Kir7.1 Inward rectifier potassium 1.50 6 0.13 3 1 0.00235 Modest apical enrichment Transporters and ion channel 13 channels j

P53985 SLC16A1, MCT1* Monocarboxylate 1.08 6 0.09 3 1 0.321 Equal abundance Transporters and ion 15 No. transporter 1 channels Q01650 SLC7A5, LAT1 Large neutral amino acids 0.909 6 0.021 5 1 0.00044 Equal abundance Transporters and ion j

transporter small subunit 1 channels 5028 Q96NY7 CLIC6 Chloride intracellular 1.59 6 0.04 23 5 7.3E-29 Modest apical enrichment Transporters and ion channel protein 6 channels Downloaded fromiovs.arvojournals.org on09/27/2021 rnpre rti oaiaini h RPE the in Localization Protein Transporter TABLE 3. Continued

P00441 SOD1 Superoxide dismutase [Cu- 3.30 6 0.29 20 4 4E-15 Mainly apical Metal ion homeostasis Zn] Q8NEW0 SLC30A7, ZNT7 Zinc transporter 7 1.65 6 0.01 3 1 5.68E-08 Modest apical enrichment Metal ion homeostasis Q9Y6M5 SLC30A1, ZNT1 Zinc transporter 1 1.44 6 0.09 8 2 0.000000171 Equal abundance Metal ion homeostasis Q9ULF5 SLC39A10, ZIP10 Zinc transporter ZIP10 0.910 6 0.052 4 1 0.0586 Equal abundance Metal ion homeostasis Q13433 SLC39A6, ZIP6 Zinc transporter ZIP6 0.950 6 0.055 3 1 0.244 Equal abundance Metal ion homeostasis Q9NY26 SLC39A1, ZIP1 Zinc transporter ZIP1 0.674 6 0.014 5 1 2.88E-09 Equal abundance Metal ion homeostasis P02792 FTL Ferritin, light chain 1.71 6 0.05 9 2 2.76E-14 Modest apical enrichment Metal ion homeostasis P02794 FTH1 Ferritin, heavy chain 1.82 6 0.13 12 3 5.25E-12 Modest apical enrichment Metal ion homeostasis P02787 TF Serotransferrin 1.16 6 0.09 4 1 0.0352 Equal abundance Metal ion homeostasis Other proteins involved in retinal well-being, or involved with AMD** O15173 PGRMC2, PMBP Membrane-associated 2.77 6 0.25 6 1 2.86E-08 Apical enrichment Photoreceptor progesterone receptor neuroprotection component 2 P98155 VLDLR Very low-density lipoprotein 1.47 6 0.06 3 1 0.000209 Equal abundance Inhibition of retinal receptor angiogenesis P36955 SERPINF1, PEDF* Pigment epithelium-derived 0.854 6 0.026 51 10 8.92E-15 Equal abundance Inhibition of angiogenesis factor and inflammation P35625 TIMP3* Metalloproteinase inhibitor 1.26 6 0.12 23 5 0.113 Equal abundance Associated with AMD, 3 Found in drusen Q92743 HTRA1 Serine protease HTRA1 0.562 6 0.02 19 3 6.92E-20 Modest basal enrichment Associated with AMD P10909 CLU Clusterin 0.566 6 0.029 5 1 0.000000212 Modest basal enrichment Found in drusen Q08380 LGALS3BP Galectin-3-binding protein 0.597 6 0.014 45 9 6.32E-52 Modest basal enrichment ECM and adhesion, Associated with AMD P02649 APOE Apolipoprotein E 0.958 6 0.024 53 10 0.00113 Equal abundance Perception of light stimulus, Drusen protein 18 * Genes encoding these proteins were identiﬁed as RPE signature genes by Strunnikova et al. IOVS † Each datum represents mean 6 SEM determined by the average of ratios of all identiﬁed peptides derived from each protein corresponding to the peptide peak areas derived from apical membrane

relative to basolateral membrane. Four independently separated membrane fractions of both apical and basolateral plasma membranes were analyzed by SWATH once (n ¼ 4). j

‡ The total number of transitions derived from identified all specific peptides (three to six transitions per peptide) was indicated. 2019 December § Data represent the identified peptide number by SWATH-MS derived from both apical and basolateral membrane. jj Localization evaluated by the expression ratios: the fold differences (apical/basolateral ratios) of 1.5-2 were considered as modest apical enrichment, 2- to 3-fold differences as apical enrichment and >3-fold differences as expression mainly on the apical surface (corresponding to apical/basolateral ratio of 0.5-0.667 as modest basal enrichment, 0.333-0.5 as basal enrichment and ratio below 0.333 as expression mainly on the basal surface). # Classification modified from categorization presented by Hongisto et al.17 ** References17,19–23 j o.60 Vol. j o 15 No. j 5029 Downloaded fromiovs.arvojournals.org on09/27/2021 rnpre rti oaiaini h RPE the in Localization Protein Transporter TABLE 4. Transporters With Drug Substrates or Potential Prodrug Targets Detected With SWATH-MS

Uniprot Apical/ Total Detected Drug Accession Protein Basolateral Transition Peptide Substrates/ Endogenous No. Abbreviation Protein Name Ratio* Localization† Number‡ Number§ P Inhibitors Substratesjj

Q15758 SLC1A5, ATB0þ Neutral amino acid 1.54 6 0.06 Modest apical 6 1 0.000000024 Potential target for Amino acids (e.g., transporter B(0) enrichment prodrugs (e.g., glutamate), thyroid aspartate- or hormones, thyroid glutamate- hormone derivatives conjugates)24 Q9Y289 SLC5A6, SMVT Sodium-dependent 1.64 6 0.11 Modest apical 6 1 0.00000158 Potential target for Vitamins, naþ multivitamin enrichment prodrugs (biotin- transporter conjugates)25 P41440 SLC19A1, RFC1 Folate transporter 1 1.18 6 0.04 Equal abundance 3 1 0.00328 Methotrexate; potential Folate, folate derivatives target for prodrugs (folate-conjugates)26 P53985 SLC16A1, MCT1 Monocarboxylate 1.08 6 0.094 Equal abundance 3 1 0.321 Salicylate, fluorescein Monocarboxylates transporter 1 valproate4,27–30 Q01650 SLC7A5, LAT1 Large neutral amino 0.909 6 0.021 Equal abundance 5 1 0.00044 L-DOPA, melphalan, Amino acids, S-nitroso-L- acids transporter gabapentin30 cysteine small subunit 1 P33527 ABCC1, MRP1 Multidrug resistance- 0.952 6 0.026 Equal abundance 35 9 0.00474 Ofloxacin, Glutathione and associated protein 1 erythromycin, glutathione clotrimazole, conjugates, cyclosporine, leukotriene c4, fluorescein, estradiol-17-beta-o- verapamil, glucuronide citalopram4,27,28 Q9HAB3 SLC52A2, RFT3 Solute carrier family 52, 0.600 6 0.017 Modest basal 5 1 4.84E-09 Potential target for Riboflavin member 2, riboflavin enrichment riboflavin analogues transporter 3 or riboflavin- conjugated prodrugs31 IOVS Q9NVC3 SLC38A7, SNAT7 Putative sodium- 0.437 6 0.032 Basal enrichment 3 1 0.0000579 Amino acids (e.g., j

coupled neutral glutamate) 2019 December amino acid transporter 7 * Each datum represents the mean 6 SEM determined by the average of ratios of all identiﬁed peptides derived from each protein corresponding to the peptide peak areas derived from apical membrane relative to basolateral membrane. Four independently separated membrane fractions of both apical and basolateral plasma membranes were analyzed by SWATH once (n ¼ 4). The proteins are presented here in descending order based on their apical/basolateral expression ratio (i.e., the higher the number the higher relative amount of protein in apical membrane and the lower the ratio the

higher the relative amount in basolateral membrane). j † Localization evaluated by the expression ratios: the fold differences (apical/basolateral ratios) of 1.5-2 were considered as modest apical enrichment, 2- to 3-fold differences as apical enrichment and 60 Vol. >3-fold differences as expression mainly on the apical surface (corresponding to apical/basolateral ratio of 0.5-0.667 as modest basal enrichment, 0.333-0.5 as basal enrichment and ratio below 0.333 as expression mainly on the basal surface). j

‡ The total number of transitions derived from identified all specific peptides (three to six transitions per peptide) was indicated. 15 No. § Data represent the identified peptide number by SWATH-MS derived from both apical and basolateral membrane. jj Substrate information was cited from The Transporter Classification Database (TCBD).32 j 5030 Transporter Protein Localization in the RPE IOVS j December 2019 j Vol. 60 j No. 15 j 5031

formation, and the marker protein expression confirm that the Detection Limit and Ion Suppression Effect in cells had properly differentiated before the membrane SWATH-MS Analysis separation. Separation of the membrane fractions appears successful. Due the sensitivity difference between QTAP and SWATH-MS, Expression of the marker proteins (LAT1, P-gp, MCT1, MCT4, only 7 transporters were detected with the SWATH-MS among MRP1) was consistent in immunolabeling and QTAP experi- the 16 transporters quantified by QTAP (Supplementary Table ments. LAT1, P-gp, and MCT1 expression were modestly S1). The transporter expression levels in apical and basolateral enriched onto the apical membrane (QTAP; Fig. 2B; Table 2; membrane fractions detected with SWATH-MS were 1.02 to immunofluorescence; Fig. 3). MCT4 was observed on all 1141 fmol/lg protein, whereas the expression levels of cellular surfaces (Fig. 3), and it was equally abundant in both nondetectable transporters were 0.197 to 3.59 fmol/lg membrane fractions (Table 2; Fig. 2B). MRP1 had equal protein. This indicates that the detection limit of SWATH-MS abundance in both membrane fractions (Table 2; Fig. 2B) and is about 1 to 3 fmol/lg protein. was observed mainly on the lateral surface via immunocyto- SWATH-MS analyses revealed the comprehensive protein chemistry (Fig. 3). This is in line with earlier literature as hfRPE expression patterns in the apical and basolateral membrane cells cultured on transwells for 4 weeks showed lateral fractions of hfRPE. The quantitative value of SWATH-MS was localization of MRP1.34 Because laterally located MRP1 had validated by comparing the result with QTAP results (Supple- similar expression in both fractions (Figs. 2, 3), the amount of mentary Table S1). Our result showed that differences of lateral membrane in both apical and basolateral membrane apical/basolateral ratios of SWATH-MS and QTAP were less than fractions is similar. CD81, basally enriched in SWATH-MS data 50%. However, we cannot exclude the possibility that SWATH- (Table 3), showed basal enrichment in intact cells (Fig. 3), MS results were affected by ion suppression effects that may confirming the success of the membrane separation. Further- occur in a sample-dependent manner. Most apical/basolateral more, we detected similar NaþKþATPase enrichment ratios expression ratios from SWATH-MS analyses were lower than (2.4- to 3.3-fold; calculated from the values in Table 2) as with those obtained by QTAP (Supplementary Table S1), suggesting that ion suppression effects take place in apical membrane. previously established plasma membrane isolation method.7 As Thus, the expression ratios (apical/basolateral) indicated in NaþKþATPase is also present in melanosomes, and therefore Tables 3 and 4 and in Supplementary Tables S5 to S8 are plasma membrane/whole cell lysate enrichment remains possibly underestimated. relatively low; we also compared the enrichment between the basolateral plasma membrane and crude basolateral membrane. This ratio (2.9-fold enrichment, calculated from RPE Surface Proteins and Ocular Supplementary Table S3) was similar as with purified plasma Pharmacokinetics membrane compared with crude membrane fraction enrich- ABC Efflux Transporters. In the current study, MRP1 ment (2.1-fold, from reference 7). All in all, our fractionation showed the highest abundance on the RPE cell surface among method produces plasma membrane fractions that have similar ABC transporters (Table 2). In vivo studies in rodents37–39 and level of membrane enrichment as the previously published a recent positron emission tomography (PET)-based study in plasma membrane isolation method used widely in the 40 7,35 humans indicated that MRPs had similar impact on the drug proteomics field. 39 þ þ uptake to the brain and retina in rats, whereas P-gp had a Na K ATPase is often enriched to the apical plasma more modest role in the transport across the blood–retinal membrane in hfRPE cultures, and this enrichment is sometimes barrier. Overall, MRPs are suggested to have stronger impact on suggested to indicate proper RPE maturity and polarization. the ocular pharmacokinetics than P-gp, and our expression However, we found equal levels of proteins expressed in both data support this conclusion as P-gp expression in the RPE was plasma membrane fractions with QTAP and SWATH-MS (Fig. lower than the expression of MRP1 and MRP5 (Table 2). 2B; Table 3). Our result is similar with earlier findings showing Most of the quantitated transporters displayed nonpolarized NaþKþATPase expression on both cellular surfaces in cultured 33 expression in the RPE, and the three apically enriched proteins human adult RPE cells with immunocytochemistry. In native (MCT1, LAT1, and P-gp) were detected and quantitated in both human RPE, NaþKþATPase is actually expressed on both cell 36 þ þ membrane fractions (Table 2; Fig. 2B). This finding seems surfaces. Na K ATPase helps to maintain ideal ion concen- different from many other tissues, where the polarized drug tration in the subretinal space, and it is vital for the functions of 41 þ transporter localization has been reported. Importantly, the the RPE and photoreceptors. Another important protein for K efflux proteins are not always expressed in a polarized manner. concentration maintenance, Kir7.1, was apically enriched 36 For instance, MRP5 did not show enrichment into luminal or (Table 3), as indicated in the earlier literature. abluminal membranes in porcine brain capillaries,42 which is Most of the detected proteins were expressed in a similar to our finding regarding the MRP5 in the hfRPE cells nonpolarized manner in hfRPE cells (Fig. 2C). This result is (Table 2). Furthermore, P-gp was detected on both sides of surprising due the well-described polarity of the human RPE human brain capillary endothelial cells with approximately 1.4- 1,2 in the literature, but it can partly be explained by the fold enrichment onto abluminal membrane.43 As emphasized underestimation of apical/basolateral expression ratios earlier, the previous literature regarding most RPE transporters caused by ion suppression in SWATH-MS and the presence has not been able to conclude their consistent localization of the lateral membrane in both membrane fractions. either on the basal or apical membrane.5 However, because P- Because our cell model consists of fetal cells, it cannot be gp was detected on both sides of human RPE tissue,44 the P-gp assumed that they fully resemble the adult RPE in vivo, even expression herein at both hfRPE membrane fractions is though polarity of the marker proteins was evident consistent with real human tissue and similar to the according to the immunofluorescence imaging (Fig. 3). enrichment in human blood–brain barrier.43 Kennedy et al.44 Furthermore, this is the first study evaluating the RPE suggested that RPE’s basal P-gp function would include polarization with quantitated protein expression, making a elimination of metabolites formed in the subretinal space and direct comparison to earlier RPE literature difficult. Due to restrict the xenobiotic entry from the choroidal blood stream. the poor tissue availability of adult human RPE tissue, the Because the protein was localized on both cell surfaces in differentiated hfRPE cells are the available material to study native noninduced tissue, apically located P-gp was suggested the proteome in the human RPE. to participate in the transport functions of RPE by delivering

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FIGURE 4. Localization of transporter proteins with drug substrates in the hfRPE cells. In the case of enrichment, the protein is illustrated with an oval shape instead of a circle. White background indicates the transporter with clinical drugs as substrates, whereas yellow background shows the proteins (SMVT, ATB0þ, TAUT, SNAT7, RFT3) that are potential targets for prodrugs. Orange color indicates proteins that can be targets for both clinical drugs and prodrugs (LAT1, RFC1).

bioactive lipids, steroids, and retinoids into the subretinal lower the RPE drug exposure after intravitreal or systemic space. P-gp is unlikely to function in an opposite direction as administration regardless their localization. Our current finding an influx pump removing substrates from the subretinal space describing the efflux protein expression on both cell surfaces into the RPE. In contrast, immunofluorescent images indicated indicates that drug exposure can be reduced by these proteins, mainly apical expression of MRP1, MRP4, and MRP5 in even further highlighting the importance of avoiding efflux cultured stem cell–derived RPE cells (hESC-RPE),45 whereas protein substrates in drug discovery toward targets in the RPE. we found these proteins to be expressed at equal levels in the SLC Transporters. Our data show that many SLC family hfRPE plasma membrane fractions (Table 2). On the other proteins (e.g., MATE1, TAUT, SMVT, LAT1, MCT1, MCT3, hand, fluorescein transport assays with porcine RPE suggested MCT4) are also present on both RPE surfaces (Tables 2, 3). The basal rather than apical enrichment of MRP proteins (i.e., clear finding is interesting from the drug delivery point of view, directional apical-to-basolateral permeation),46 but transport of because influx proteins may enable drug delivery to the RPE or fluorescein may be affected also by OCTs and not only MRPs. neural retina. Lactate, which is a byproduct of photoreceptor Because many substrates overlap between efflux and influx energy metabolism, is removed from the subretinal space into proteins, firm conclusions on the function of a specific the systemic circulation via monocarboxylate transporters transporter are difficult to reach. The physiologic role of the (MCT1 and MCT3), but these transporters are also important highly abundant MRP1 in the RPE involves maintenance of in pharmacokinetics, because MCTs can transport drugs, such cellular thiol homeostasis and participation in efflux of cellular as valproate (Table 4). Analogously, LAT1 and TAUT have been glutathione.34 under investigation as potential pathways for CNS drug Our findings suggest that drug permeation across the RPE targeting.26,49 Also, SMVT and sodium- and chloride-dependent may not display strong directionality, because only modest neutral and basic amino acid transporter (ATB0þ) have been differences were found between their expression on RPE proposed as avenues for drug targeting; both displayed modest apical and basolateral membranes (Fig. 2B; Table 2). As the enrichment onto the apical membrane of hfRPE cells. permeation of transporter protein substrates across primary Furthermore, basolaterally enriched glutamate transporter RPE cultures has not been reported,6 functionality data are putative sodium-coupled neutral amino acid transporter 7 needed. The nonpolarized expression pattern of transporters (SNAT7) (Table 4) has been suggested to enhance blood–brain is, however, consistent with QSPR models describing the drug barrier permeation of glutamate conjugates in rodents.50 kinetics in the eye after intravitreal injection47 or systemic However, the transporter-mediated drug targeting to the retina administration.48 The models, based on simple physical– has not been proven, and further investigations are needed. chemical descriptors for passive diffusion, were able to Summary. The protein enrichment values we present in describe and predict drug permeation between the vitreous this paper reveal the localization of the detected proteins and blood circulation for large compound sets without among RPE surfaces. The summary of our main findings significant outliers. This suggests that strong transport regarding the drug transporter localization in hfRPE cells is directionality was absent in those data, and permeation was illustrated in Figure 4. As stated previously, most of the drug dominated by passive diffusion. However, as the RPE itself is an transporting proteins localize on both cellular surfaces. important drug target, we recently evaluated the potential Together with the previous literature,5,47,48 our findings influence of efflux proteins for the drug exposure in the RPE.5 suggest that the transporters on the RPE surface have higher Our simulations showed that efflux proteins can significantly impact on RPE drug exposure than on the permeation across

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the outer blood–retinal barrier. The transporter quantification 8. Huang Q, Yang L, Luo J, et al. SWATH enables precise label- and localization data of this report can be further used in free quantification on proteome scale. Proteomics. 2015;15: building pharmacokinetic simulation models for the posterior 1215–1223. eye segment. 9. Nakamura K, Hirayama-Kurogi M, Ito S, et al. Large-scale multiplex absolute protein quantification of drug-metaboliz- ing enzymes and transporters in human intestine, liver, and CONCLUSIONS kidney microsomes by SWATH-MS: comparison with MRM/ SRM and HR-MRM/PRM. Proteomics. 2016;16:2106–2117. In this paper, we provide information of 1201 proteins present 10. Miyauchi E, Furuta T, Ohtsuki S, et al. Identification of blood on hfRPE plasma membrane fractions (Supplementary Tables biomarkers in glioblastoma by SWATH mass spectrometry and S5–S8). The identification and the quantitative expression quantitative targeted absolute proteomics. PLoS One. 2018; ratios of these proteins are valuable in ocular research as they 13:e0193799. can increase the understanding of the outer blood–retinal 11. Gillet LC, Navarro P, Tate S, et al. Targeted data extraction of barrier functions. Our study reveals relatively nonpolarized the MS/MS spectra generated by data-independent acquisi- localization of transporters in human primary RPE cells. This tion: a new concept for consistent and accurate proteome finding highlights the need of avoiding efflux protein analysis. Mol Cell Proteomics. 2012;11:O111.016717. substrates in retinal drug discovery, especially when the RPE 12. Adijanto J, Philp NJ. Cultured primary human fetal retinal is the target tissue. We provide the abundances of 15 pigment epithelium (hfRPE) as a model for evaluating RPE transporter proteins scaled to RPE tissue level, which may be metabolism. Exp Eye Res. 2014;126:77–84. further used in pharmacokinetic model building. Because 13. Fong-ngern K, Chiangjong W, Thongboonkerd V. Peeling as a hfRPE cell cultures are vital for RPE research, we predict that novel, simple, and effective method for isolation of apical the membrane proteomes presented here might also be used as membrane from intact polarized epithelial cells. Anal a tool to explain possible differences in the observed in vitro Biochem. 2009;395:25–32. and in vivo RPE functions. 14. Uchida Y, Tachikawa M, Obuchi W, et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/ Acknowledgments MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker The authors thank laboratory technician Lea Pirskanen for valuable proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J help in the hfRPE cell culture and fractionation and Anna mice. Fluids Barriers CNS. 2013;10:21. Poutiainen, MSc, for important contributions in the membrane 15. 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