Optimized Quantitative Cell Surface Protein Biotinylation Protocol

Introduction

Accurate prediction of in vivo drug clearance from in vitro studies (IVIVE) is important in drug discovery and development to estimate first in human dose, drug-drug interactions, drug tissue concentrations and thus the efficacy and toxicity liability of a new chemical entity.  Transporters play an important role in drug disposition.  However, unlike drugs cleared by metabolism, IVIVE of transporter-mediated clearance of drugs has been challenging (1, 2).  Although recent studies have shown some success (3, 4), most studies conducted in human hepatocytes have shown considerable underprediction of the observed in vivo transporter-mediated clearance (5, 6).  One possible explanation for this underprediction is that transporter expression in human hepatocytes (total or that expressed in the plasma membrane) does not reflect that in human liver tissue (7).  We and others have hypothesized that transporter-mediated clearance of drugs can also be predicted based on transport activity and expression in cells individually expressing the relevant transporters (3, 4, 8).  For either approach (based on human hepatocytes or cells) to be successful, one should ideally quantify the expression of transporter proteins in the plasma membrane of transporter-expressing cells or human hepatocytes and scale those expression levels to the expression in human liver tissue.

There are several lines of evidence that hepatic transporters may not be exclusively localized in the plasma membrane of human hepatocytes or transporter-expressing cells. In cryopreserved human hepatocytes, a large fraction of total OATP1B1 and OATP1B3 expression is intracellular (9).  NTCP is sequestered within intracellular vesicular “reservoir” compartments in HepG2 cells and isolated rat hepatocytes (10).  Canalicular efflux transporters, P-gp and Mrp2, are rapidly internalized after isolation of rat hepatocytes (11).  As a result, quantifying plasma membrane expression of drug transporters in hepatocytes or primary cells may result in more accurate IVIVE of transporter-mediated clearance. Therefore, in the present study, our objectives were:

a) to optimize a method that can be used to quantify plasma membrane protein expression of transporters in transporter-expressing cell lines or primary cells derived from human tissues (e.g. human hepatocytes).  To do so, using CHO cells expressing OATP1B1, we optimized an existing biotinylation method (12) to make it quantitative. This method uses a membrane impermeable biotinylation reagent, sulfo-NHS-SS-biotin;

b) to determine if the percent of OATP1B1 expressed in the plasma membrane was host-cell dependent (CHO vs. MDCKII vs. HEK293 cells);

c) to determine if the percent of other major sinusoidal uptake drug transporters OATP2B1, 1B3 and NTCP in plasma membrane was similar to that of OATP1B1 when these transporters are expressed in different host cells.

Cell surface biotinylation was introduced about three decades ago (12), but since then it has been mostly used for qualitative or semi-quantitative purposes. Here, we present an optimized quantitative cell surface protein biotinylation protocol that can be to be used for quantitative cell surface protein expression by LC-MS/MS proteomics. This protocol will help in the understanding of the plasma membrane transporter-mediated drug disposition and in the determination of the quantitative expression of any cell surface protein of interest in the cell lines.

Materials and Methods

Chemicals and Reagents

The ProteoExtract native membrane protein extraction kit was procured from Calbiochem (Temecula, CA). Synthetic signature peptides for OATP1B1, OATP2B1, OATP1B3, NTCP and Na⁺– K⁺ ATPase were obtained from New England Peptides (Boston, MA). The corresponding stable isotope labeled (SIL) peptides for OATP1B1, OATP2B1, OATP1B3, NTCP and Na⁺– K⁺ ATPase, protein quantification BCA assay kit, dithiothreitol (DTT), iodoacetamide (IAA), MS grade trypsin, DMEM (Dulbecco’s Modified Eagle Medium) high glucose medium (Gibco, Life Technologies), DMEM low glucose medium (Gibco, Life Technologies), DPBS (Dulbecco’s Phosphate-Buffered Saline), Hank’s Balanced Salt Solution (HBSS), and Pierce cell surface protein isolation kit were obtained from Thermo Scientific (Rockford, IL). Human albumin was procured from Calbiochem (Temecula, CA). Pierce cell surface protein isolation kit contains sulfo-NHS-SS-biotin, quenching solution (100 mM glycine), lysis buffer, neutravidin agarose gel, wash buffer, column accessory pack, DTT, phosphate buffer and Tris buffer. Composition of lysis buffer and wash buffer was not disclosed in the Pierce cell surface protein isolation kit. All reagents were analytical grade.

Transporter-expressing cell lines

OATP1B1-expressing CHO and MDCKII cells were generously provided by Dr. Bruno Stieger, University Hospital Zurich, Switzerland and Dr. Markus Keiser, University of Greifswald, Germany, respectively. OATP1B1-expressing HEK293, OATP1B3-expressing HEK293, OATP2B1-expressing MDCKII and NTCP-expressing CHO cells were generously provided by SOLVO Biotechnology, Hungary.

Cell culture 

Transporter-expressing cells: To isolate plasma membrane using  biotinylation, transporter expressing cells were grown in T-75 cm2 flasks at a density of 1.4×106 cells per flask with 20 mL of low glucose (CHO cells) or high glucose (MDCKII, HEK293 cells) DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin. Medium was changed daily. Cells were incubated at 37 °C with 5% carbon dioxide in a humidified incubator for 2 days.  Subsequently, for OATP1B1-expressing CHO and MDCKII cells, the medium was removed and replaced with DMEM medium containing 10 mM sodium butyrate for 24 hours (13).  The protocol for OTAP1B1-expressing HEK293, OATP2B1-expressing MDCKII, OATP1B3-expressing HEK293 and NTCP-expressing CHO cells did not recommend use of sodium butyrate.

Biotinylation protocol for plasma membrane isolation

The Pierce™ cell surface isolation protocol (to isolate plasma membrane) was optimized as described below (Fig. 1, 2).  Briefly, at day 3 of cell culture, when confluency had been reached, the medium was removed and cells were washed twice with 10 mL of DPBS buffer (pH 7.4) at 37 °C. The DPBS buffer was quickly replaced with 10 mL of varying concentrations of biotinylation reagent (sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate; sulfo-NHS-SS-biotin). The cells were incubated with sulfo-NHS-SS-biotin for the duration and temperature listed below, to non-specifically biotinylate primary amines (e.g. lysine or arginine) or N-terminal primary amines of extracellular peptide residues in the plasma membrane. After the indicated time, the sulfo-NHS-SS-biotin solution was removed and the biotinylation reaction was quenched with 10 mL of DPBS buffer containing 500 µL of quenching solution (100 mM glycine).

Then, cells were scraped from the flask and centrifuged at 500 xg for 3 minutes at 4 °C. The supernatant was discarded and the cell pellet was washed with 5 mL Tris saline buffer before further centrifugation at 500 xg for 3 minutes at 4 °C. After centrifugation, the supernatant was discarded and the cells were lysed in 500 µL lysis buffer (containing 2.5 µL protease inhibitor) by incubating them for 30 minutes on ice with intermittent vortexing and sonication (3 times, 5-10 seconds each, during 30 minutes incubation in ice). Sonication was done for a very short duration to ensure minimal or no inside-out membrane vesicle formation (14). The cell lysate was centrifuged at 10,000 xg at 4 °C to pellet intact nuclei and mitochondria. The supernatant of cell lysate [containing total cell membrane; sample no. 1] was collected and 375 µL of it was incubated (about 125 µL was stored at -80 °C for further proteomics analysis) at room temperature with end-over-end mixing in a column containing neutravidin resin for 1 hour.

This column was then centrifuged (1,000 xg for 1 min) and the first centrifugate containing non-biotinylated membrane was collected [sample no. 2]. The column was washed three times with 400 µL, 250 µL, and 200 µL of wash buffer containing protease inhibitor (5 µL/ mL) [sample no. 3, 4 and 5] and centrifuged (1,000 xg for 1 min) after each washing. Each time, the centrifugate (also containing the non-biotinylated membrane) was collected and volume was measured accurately and stored at -80 °C for further proteomics analysis.  Then, the biotinylated plasma membrane was eluted from the neutravidin resin column by incubating the column with 400 µL of elution buffer [sample no. 6], containing 50 mM DTT. DTT reduces the disulfide bonds, between the extracellular lysine or arginine residues and biotin, thus allowing the plasma membrane to elute.

Then, the neutravidin resin column was washed twice with 250 µL and 200 µL of wash buffer [sample no. 7 and 8; also containing the plasma membrane] containing protease inhibitor (5 µL/ mL) followed by centrifugation (1,000 xg for 1 min). The centrifugate, containing the plasma membrane, was collected and volume was measured accurately and stored at -80 °C for further proteomics analysis.

To ensure that biotinylated plasma membrane was not lost in sample 2, 250 µL of this centrifugate was incubated with another neutravidin resin column for 1 hour at room temperature. The column was centrifuged at 1,000 xg for 1 min [sample no. 9] then washed with 400 µL of wash buffer [sample no. 10] containing protease inhibitor (5 µL/ mL) and centrifuged (1,000 xg for 1 min).  The centrifugate (containing non-biotinylated membrane) was collected and volume was measured accurately and stored at -80 °C for further proteomics analysis.  The biotinylated plasma membrane was eluted from 2^nd neutravidin resin column [sample no. 11] by centrifugation (1,000 xg for 1 min) and washed with 250 µL of wash buffer [sample no. 12]. Sample no. 1 contained the total cell membrane. The biotinylated plasma membrane was assumed to be contained in sample no. 6, 7, 8, 11 and 12 and the non-biotinylated membrane were assumed to be contained in sample no. 2, 3, 4 and 5.  Recovery of Na⁺– K⁺ ATPase was used as a marker of plasma membrane.

Optimization of biotinylation experimental conditions

1. Optimization of biotin reagent (sulfo-NHS-SS-biotin) concentration for plasma membrane isolation from OATP1B1-expressing plated CHO cells (4 °C)

OATP1B1-expressing CHO cells were plated at a cell density of 1.4×10⁶ cells/ T-75 cm² flask. Two flasks were used for each concentration of sulfo-NHS-SS-biotin reagent. Once the cells were confluent in three days, they were incubated with various concentrations (0.24 mg/mL, 0.78 mg/mL, 1.02 mg/mL, 1.26 mg/mL, 1.5 mg/mL, 1.98 mg/mL or 2.46 mg/mL) of the membrane impermeable biotinylation reagent (sulfo-NHS-SS-biotin) for one hr at 4 °C. Then, the percent of OATP1B1, Na⁺– K⁺ ATPase and calreticulin (an endoplasmic reticulum (ER) marker) (15) in the biotinylated plasma membrane fractions, at each sulfo-NHS-SS-biotin concentration, was compared.

2. Optimization of temperature of incubation with sulfo-NHS-SS-biotin  for plasma membrane isolation from OATP1B1-expressing plated CHO cells

To study the effect of incubation temperature on isolation of plasma membrane, biotinylation of cells was conducted at two different temperatures (4 °C and 37 °C) with plated OATP1B1-expressing cells. At day 3 of cell culture, OATP1B1-expressing CHO cells were incubated with 1.50 mg/mL of sulfo-NHS-SS-biotin for 1 hour at 4 °C and 37 °C. Then, the percent of OATP1B1, Na⁺– K⁺ ATPase and calreticulin in the biotinylated plasma membrane fraction was compared between 4 °C and 37 °C.

3. Optimization of biotin reagent (sulfo-NHS-SS-biotin) concentration for plasma membrane isolation from OATP1B1-expressing plated CHO cells (37 °C)

OATP1B1-expressing CHO cells were plated at a cell density of 1.4×10⁶ cells/ T-75 cm² flask. Two flasks were used for each concentration of sulfo-NHS-SS-biotin reagent. OATP1B1-expressing CHO cells at day 3 of cell culture were incubated with different concentrations (0.24 mg/mL, 0.78 mg/mL, 1.02 mg/mL, 1.26 mg/mL, 1.5 mg/mL, 1.98 mg/mL or 2.46 mg/mL) of sulfo-NHS-SS-biotin for an hour at 37 °C. The percent of OATP1B1, Na⁺– K⁺ ATPase and calreticulin in the biotinylated plasma membrane was compared at various concentrations of sulfo-NHS-SS-biotin.

The above optimized biotinylation method was used to determine plasma membrane protein expression of OATP1B1 in OATP1B1-expressing CHO, MDCKII or HEK293 cells, OATP2B1 in OATP2B1-expressing MDCKII cells, OATP1B3 in OATP1B3-expressing HEK293 cells, and NTCP in NTCP-expressing CHO cells.

Sample preparation for LC-MS/MS proteomics

The sample preparation method was as described previously (16). Twenty microliters of 2.0 mg/mL (or lower protein concentration) of the transporter expressing cell line or samples obtained through biotinylation experiments were mixed with 18 μL of 3% sodium deoxycholate (w/v), 6 μL DTT (100 mM), 15 μL of ammonium bicarbonate buffer (50 mM, pH 7.8) and 10 μL of human albumin (10 mg/mL).  After incubation at 95 °C for 5 minutes, followed by cooling at room temperature, 6 μL of iodoacetamide (200 mM; an alkylating agent) were added to the mixture, followed by incubation at room temperature for 20 minutes in the dark. To concentrate the sample, ice-cold methanol (0.5 mL), chloroform (0.2 mL), and water (0.45 mL) were added to each sample. After centrifugation at 4 °C for 8 minutes at 13,000 xg, the pellet was washed once with ice-cold methanol (1.0 mL) and was dissolved in 55 μL of reconstituted solution (a mixture of 15 μL sodium deoxycholate 3% (w/v) and 40 μL ammonium bicarbonate buffer). Finally, the protein sample was digested with 20 μL of trypsin. Protein to trypsin ratio was 25:1 (w/w). After the 24-hour incubation at 37 °C, the digestion reaction was quenched by 30 mL of labeled peptide internal standard (SIL) cocktail (prepared in 80% acetonitrile in water containing 0.1% formic acid; final concentration of SIL: 0.1–0.25 nM). The samples were centrifuged at 5000 xg for 5 minutes at 4 °C, and 5 μL of the supernatant were introduced into the LC-MS/MS system.

LC-MS/MS quantification of transporters and marker proteins

The protein expression of OATP1B1, OATP2B1, OATP1B3, NTCP, plasma membrane marker (Na⁺– K⁺ ATPase) or ER marker (calreticulin) in cell lines were quantified using LC-MS/MS and surrogate peptides as described before (13, 17) (Table 1). The chosen surrogate peptides were peptide sequences in the intracellular domain of the transporters and therefore should not be modified by the membrane impermeable sulfo-NHS-SS-biotin provided the transporters are predominantly present in the plasma membrane. The corresponding heavy labeled peptide was used as an internal standard. The MRM parameters for quantification of the subcellular marker proteins are shown in Table 1.  Total protein concentration in individual samples was determined using the BCA protein assay kit (Thermo Fisher Scientific).

Analytical method parameters

AB Sciex 6500 triple-quadrupole mass spectrometer (Sciex, Framingham, MA, U.S.A.) coupled to the Water Acquity UPLC system (Waters Corporation, Milford, MA) operated in electrospray positive ionization mode was used for liquid chromatography-tandem mass spectroscopy (LC-MS/ MS) analysis of the signature peptides. The doubly charged parent to singly charged product transitions for the analyte peptides and their respective SIL peptides were monitored using optimized LC-MS/MS parameters (Table 1). The signature peptides and SIL peptide internal standards were separated on an Acquity UPLC HSS T3 Column, (1.8 µm, 2.1 mm X 100 mm) with a 0.2 µm Inlet frits (Waters, Milford, MA, USA). Mobile phases (0.3 mL/min) consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program for UPLC method for determination of OATP1B1,  Na⁺– K⁺ ATPase  and calreticulin in OATP1B1-expressing CHO/MDCKII/HEK293 cells in biotinylation study was: 0–2 minutes: 3% B; 2–9 minutes: 3–50% B; 9–9.5 minutes: 50–90% B; 9.5–10.5 minutes: 90% B; 10.5–11 minutes: 90–3%B; 11–14 minutes: 3–3%B including washing and re-equilibration for 3 minutes. The gradient program for UPLC method for determination of OATP1B3, OATP2B1, NTCP, Na⁺– K⁺ ATPase  and calreticulin in OATP1B3 or OATP2B1 or NTCP expressing cells was: 0–2 minutes: 3% B; 2–20 minutes: 3–45% B; 20–23 minutes: 45–90% B; 23–24 minutes: 90% B; 24–24.5 minutes: 90–3%B; 24.5–28 minutes: 3–3%B including washing and re-equilibration for 3.5 minutes.

Data analysis

Calculating percent of transporters expressed in the plasma membrane

A single biotinylation experiment yielded 12 samples including the lysate, biotinylated, non-biotinylated and wash fractions (Fig. 2). The volume of every sample was measured accurately. The expression of OATP1B1, OATP1B3, OATP2B1, NTCP, Na⁺– K⁺ ATPase and calreticulin was quantified in the lysate, non-biotinylated membrane, biotinylated membrane and various associated wash fractions using LC-MS/MS. Unique stable labeled internal standards peptides of OATP1B1 or OATP1B3 or OATP2B1 or NTCP, and Na⁺/K⁺-ATPase were used to determine the area ratio in the samples obtained through biotinylation experiment. For calreticulin, stable labeled internal standard was not available (Table 1) so, calreticulin area ratio was determined using the stable labeled internal standard of Na⁺– K⁺-ATPase. Samples containing biotinylated fraction had DTT (reducing agent), which interfered with protein estimation by BCA assay, so activity/expression could not be normalized to total protein. The aim of this study was to estimate the fraction of transporter expressed in the plasma membrane compared to the total cell expression. So, determination of the absolute transporter expression was not necessary. Therefore, for each sample from the biotinylation experiment, the area ratio (analyte/internal standard area ratio) was multiplied by the volume of the sample and the obtained value was used for calculation of percent transporter expression in the plasma membrane (equation 1) and total protein recovery (equation 2). The recovery of each protein was calculated as total recovered in biotinylated plus non-biotinylated fractions expressed as a percent of that in the lysate.

Equation 1.

Biotinylation %=∑area ratio ×volume µL of sample 6,7 and 8Area ratio of sample no.1×375 µL×100+∑area ratio ×volume µL of sample 11 and 12Area ratio of sample no.2×250 µL×100

Equation 2.

Recovery %=∑area ratio ×volume (µL) ofsample 2, 3, 4, 5, 6,7 and 8 Area ratio of sample no.1×375 µL×100

Statistical Analysis

Mann–Whitney U test was performed to test the statistical difference between plasma membrane expression of OATP1B1 in OATP1B1-expressing CHO, MDCKII cells and HEK293 cells.

Results

Optimization of biotinylation conditions to isolate plasma membrane from OATP1B1-expressing CHO cells

The effect of sulfo-NHS-SS-biotin concentration, at 4 °C, on the yield of plasma membrane from OATP1B1 expressing cells:   The recovery of OATP1B1 and Na⁺– K⁺ ATPase in the biotinylated plasma membrane fraction was markedly increased with increasing sulfo-NHS-SS-biotin concentration and appeared to plateau at 1.5 mg/mL of sulfo-NHS-SS-biotin (Fig. 3).  Negligible presence of the intracellular protein, calreticulin, was observed in the plasma membrane fraction at all concentrations of sulfo-NHS-SS-biotin. Mass balance showed that the recovery of all three proteins was 87.5-115.5% irrespective of the sulfo-NHS-SS-biotin concentration.

Effect of incubation temperature (37 °C vs. 4 °C) on the yield of plasma membrane isolated from OATP1B1-expressing CHO cells at sulfo-NHS-SS-biotin concentration of 1.5 mg/mL: There was a substantial increase in biotinylation of plasma membrane at 37 °C vs. 4 °C, which was reflected in increased yield of OATP1B1 (80.2 % versus 53.5%) and Na⁺– K⁺ ATPase (90.4 % versus 58.0%) in the biotinylated fraction (data not shown).  The corresponding recovery of calreticulin remained <2.5%.  Mass balance showed that the recovery of all three proteins was 92.1-102.3%.  Therefore, the incubation temperature of 37 °C was used in all subsequent biotinylation studies.

Optimization of sulfo-NHS-SS-biotin concentration, at 37 °C , to maximize the yield of plasma membrane from OATP1B1-expressing CHO cells:   The yield of plasma membrane, as measured by recovery of OATP1B1 (94.1%) and Na⁺– K⁺ ATPase (84.0%) in the biotinylated fraction, was maximal at 0.78 mg/mL of sulfo-NHS-SS-biotin (Fig. 4).  The corresponding yield of the intracellular protein calreticulin remained negligible (<2%).  Mass balance studies showed that the recovery of all three proteins was 101.8-117.2%.  Consequently, 0.78 mg/mL of sulfo-NHS-SS-biotin and 37 °C was used in all subsequent biotinylation studies described below to determine the percent of a transporter expressed in the plasma membrane of cells expressing the transporter of interest.

Quantification of percent of transporter expressed in plasma membrane of OATP1B1 expressed in CHO, MDCKII or HEK293 cells, OATP2B1 expressed in MDCKII cells, OATP1B3 expressed in HEK293 cells, and NTCP expressed in CHO cells, using the optimized biotinylation protocol.

Mean (± SD) Percent of OATP1B1 expressed in the plasma membrane of CHO, MDCKII and HEK293 cells was 79.7% (±4.7%), 67.7% (±12.2%) and 65.3 (±6.8%) respectively (Fig. 5).  The percent of plasma membrane expression of OATP1B1 in MDCK or HEK293 was significantly lower than that in CHO cells.  The percent of plasma membrane expression of OATP1B3 in HEK293 cells, OATP2B1 in MDCKII cells and NTCP in CHO cells was 63.2% (±1.6 %), 37.1% (±15.7%) and 71.7% (±1.2%) respectively (Fig. 6).

Discussion

For successful IVIVE of transporter-based clearance, it is important to quantify the protein expression of the transporter in the plasma membrane of cells (transfected or primary cells) vs. the expression of the transporter in total cell membrane. If the latter does not equal the former, IVIVE of pharmacokinetics of a drug based on the latter will lead to incorrect prediction of the plasma clearance and in vivo plasma and tissue concentrations of the drug.  For IVIVE of the transporter-mediated clearance of a new chemical entity, prediction of clearance of the new chemical entity within ~2-fold of its ultimately determined clearance in a Phase 1 study would be acceptable,  Therefore, we were most interested in whether plasma membrane expression of the investigated hepatic uptake transporters was less than 75% of its total expression in the cells.   When such plasma membrane protein expression was greater than 75% of the total expression,  the need to add a correction factor (i.e. percent expressed in the plasma membrane) for IVIVE of transporter-mediated clearance would be less compelling .

Here we report optimization of a biotinylation method for quantitative determination of the percent of a transporter protein expressed in the plasma membrane compared to its total cell expression.  To conduct this optimization, we began with the biotinylation method recommended by the manufacturer (18).  Cell surface biotinylation method (or similar methods) has been previously used by others (19-21) for relative quantification of plasma membrane expression of membrane proteins in cells, especially in studies where the protein has been genetically modified (22-24).  To our knowledge, the manufacturer’s method has been adopted by researchers without examining the optimal conditions for its application.  Therefore, we optimized the method and coupled it to LC-MS/MS proteomics for absolute quantification of plasma membrane protein of interest as well as Na⁺/K⁺-ATPase (a plasma membrane marker protein) and calreticulin (an ER marker protein).  The latter allowed us to confirm that the biotinylation reagent was indeed “plasma membrane impermeable”.   In addition, through mass balance, we ensured that our method resulted in complete recovery of all the proteins quantified.   Where this recovery was not within 100±25% of the total protein expression in the cell homogenate, the data were discarded.

We found that the optimized biotinylation protocol (0.78 mg/mL of sulfo-NHS-SS-biotin incubated for 1 hr with OATP1B1 expressing CHO cells at 37 °C) resulted in maximum yield of plasma membrane as measured by percent of OATP1B1 (94.1%) and Na⁺– K⁺ ATPase (84.0%) in biotinylated plasma membrane and negligible presence (<12%) of the intracellular protein, calreticulin,  in the biotinylated fraction (Fig. 4).  Loder et al. also found that plasma membrane expression of the dopamine transporter, in stably transfected PC12 cells, was greater at 37 °C (physiologically relevant  temperature) than 4 °C in a biotinylation study (19).  They concluded that at 4 °C, the dopamine transporter was internalized.  While that may also be true for OATP1B1, it is also possible that the biotinylation reaction was incomplete at 4 °C.  But the latter hypothesis can be discounted as longer incubation times (> 2 hr) at 4 °C did not result in increased yield of the plasma membrane (data not shown). In all these experiments, the presence of calreticulin in the biotinylated plasma membrane fraction was minimal, indicating little intracellular penetration of the biotinylation reagent.   Moreover, the percent of Na⁺– K⁺ ATPase in the plasma membrane closely tracked the percent of transporter expressed in the membrane.  The optimal biotinylation conditions described here were confirmed in an another cell line (HEK293 cells; data not shown).

Next, we determined if the percent of OATP1B1 in the plasma membrane was dependent on the host cell-line.  A survey of the literature indicated that researchers typically use CHO, HEK293 or MDCKII cells expressing OATP1B1.  Our data show that OATP1B1 is predominantly expressed in the plasma membrane of all these cell lines (Fig. 5).  The presence of calreticulin in the plasma membrane vs. total membrane fraction was less than 10.5%, indicated high purity of the plasma membrane preparation.  Although the percent of OATP1B1 expressed in the plasma membrane of CHO cells was significantly higher than that in MDCKII and HEK293 cells, we do not consider this difference to be biologically significant.  Though the biotinylation studies with OATP1B1-expressing CHO and MDCKII cells were conducted over a one year period, the fraction of OATP1B1 expressed in plasma membrane to the total expression remained consistent over time.

Next, we determined the percent of OATP1B3, OATP2B1 and NTCP expressed in the plasma membrane when these transporters were expressed in HEK293, MDCKII or CHO cells respectively.  These host cell lines (CHO, HEK293 and MDCKII) were used as they were the cells that were readily available to us and are frequently used by various pharmaceutical companies.  The majority of OATP1B3 and NTCP were found to be expressed in the plasma membrane of these cells (Fig. 6).  However, only 37% of OATP2B1 was expressed in the plasma membrane of MDCKII cells. It has been shown through immunofluorescence microscopy study in human liver that OATP1B1, OATP2B1, and OATP1B3 are predominantly localized at the basolateral membrane and and MRP2 is predominately located at the canalicular membrane (25).  Therefore, IVIVE of transporter-mediated clearance based on their expressions in cell lines will need a scaling factor based on the percent of the transporter expressed in the plasma membrane of these cells.

For drugs predominantly eliminated unchanged in the feces by the liver, the gold standard in vitro model to predict hepatobiliary clearance is sandwich culture human hepatocytes (SCHH). Yet, this in vitromodel is well reported to underpredict transporter mediated hepatic uptake clearance of drugs (6). One proposed reason for this observation is that plasma or total transporter expression in SCHH differs from in vivo liver tissue (26).  However, determination of plasma membrane expression of transporters in liver tissues, using biotinylation, is not possible without isolating hepatocytes.  This is because homogenization of tissue to determine transporter expression will result in rupture of the cells.   Therefore, a solution for this  problem is to assume that transporter expression in plasma membrane immediately after isolation of the cells (i.e. primary cells) represents the plasma membrane expression of the transporter in the corresponding tissue. This plasma membrane isolation technique through cell surface biotinylation is applicable to all mentioned cell lines (CHO, HEK293 and MDCKII) irrespective of the transporter expression level. The limiting step could be LC-MS/MS sensitivity in the case of no or very low expression of the transporter.   It will be interesting to see if transporter-expression-based IVIVE of drug disposition based on such primary cells (e.g. cryopreserved human hepatocytes) is successful after determination of percent of transporter expressed in the plasma membrane.

In summary, we have described an optimized biotinylation method to quantify the plasma membrane expression of transporters (or for that matter any protein) in cell lines. This protocol should also be applicable to quantify plasma membrane expression of transporters in primary cells (e.g. human hepatocytes).  In the future, it will be important to apply this technique to determine if plasma membrane expression of drug transporters, other than OATP1B1, is independent of the host cell line.   In addition, before others use the data presented above for the various transporters and cell lines, it will be important to demonstrate that these values are invariant across laboratories.   Moreover, the optimized biotinylation technique presented here has utility beyond IVIVE of transporter-based clearance.  This optimized biotinylation protocol could be used in the future to understand the impact of post-translational modifications, polymorphism, disease and drugs on trafficking of proteins to the plasma membrane.

Acknowledgements:

The authors thank Dr. Bruno Stieger, University Hospital Zurich, for OATP1B1-expressing CHO cells, Dr. Markus Keiser, University of Greifswald for OATP1B1-expressing MDCKII cells, Solvo Biotechnology for OATP1B1-expressing HEK293, OATP1B3-expressing HEK293, OATP2B1-expressing MDCKII and NTCP-expressing CHO cells. The authors thank Dr. Sarah Billington for her help in reviewing this manuscript. Vineet Kumar was supported by the Simcyp Grant & Partnership Scheme (Certera).

Authorship Contributions

Participated in research design:  Kumar, Nguyen, Unadkat.

Conducted experiments: Kumar, Nguyen.

Contributed new reagents or analytic tools: Tóth, Juhasz

Performed data analysis: Kumar, Nguyen, Unadkat.

Wrote or contributed to the writing of the manuscript: Kumar, Unadkat.

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