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Development of a highly sensitive method using LC-MRM to quantify
membrane P-glycoprotein in biological matrices and relationship to
transport function
Tasso Miliotis, Liaqat Ali, Johan E Palm, Anders J Lundqvist, Martin Ahnoff,
Tommy B Andersson, and Constanze Hilgendorf
AstraZeneca R&D, Innovative Medicines, Mölndal, Sweden
DMD Fast Forward. Published on September 23, 2011 as doi:10.1124/dmd.111.040774
Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title:
LC-MRM quantification P-gp
Corresponding Author:
Constanze Hilgendorf, PhD
AstraZeneca R&D Mölndal
S-431 83 Mölndal
Phone +46 31 706 5349
Fax +46 31 776 3786
E-mail: [email protected]
Number of text pages: 38
Number of tables: 3
Number of figures: 6
Number of references: 23
Number of words in the Abstract: 226
Number of words in the Introduction: 735
Number of words in the Discussion: 1372
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Abbreviations
ABC, ATP binding cassette; AMBIC, ammonium bicarbonate; BCRP, breast cancer
resistance protein, ABCG2; BSEP, bile salt export pump; DMEM, Dulbecco’s modified
Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionisation;
HBSS, Hanks’ Balanced Salt Solution; HEK, human embryonic kidney; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; IAA, iodoacetamide; LC, liquid
chromatography; MRM, multiple reaction monitoring; MRP2, multidrug resistance associated
protein 2, ABCC2; NMQ, N-methylquinidine; PBS, phosphate-buffered saline; PPS, 3-[3-
(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate; RT-PCR, real time polymerase chain
reaction; PEI, polyethyleneimine; P-gp, permeability glycoprotein, ABCB1, MDR1; SDS,
sodium dodecyl sulfate
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Abstract
The quantification of P-gp (P-glycoprotein, ABCB1, MDR1) protein in biological matrices is
considered a key factor missing for useful translation of in vitro functional data to the in vivo
situation and for comparison of transporter data between different in-vitro models. In the
present study an LC-MS method was developed to quantify P-gp membrane protein levels in
different biological matrices. The amount of P-gp transporter protein was measured in Caco-2
cell monolayers and in inside-out HEK-MDR1 vesicles. From both in vitro systems two
preparations with different functionality were used. Transporter-function was determined as
digoxin efflux in Caco-2 cell monolayers and N-methylquinidine uptake in membrane
vesicles, additionally mRNA expression in the Caco-2 monolayers was measured. The results
showed an excellent relationship between NMQ-uptake functionality in inside-out HEK-
MDR1 vesicles and protein contents. Similar concordance between the digoxin efflux and
P-gp content in different Caco-2 cell cultures was observed, while mRNA levels are
indicative of increased P-gp content and activity in older Caco-2 cultures, however not
yielding the same quantitative relationship. The results from both Caco-2 and HEK-MDR1
membrane vesicles confirm that the protein content is directly related to the level of activity in
the respective system. The presented method to quantify P-gp protein by LC-MRM will
facilitate the development of future methodologies to bridge between expression systems and
cell/tissue models and to scale from in-vitro models to whole organs.
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Introduction
Drug transporters are increasingly recognized as important for drug disposition and drug-drug
interactions. P-gp (P-glycoprotein, ABCB1, MDR1) is a key transporter active on many drugs
and there is a large body of clinical evidence of its critical role in drug absorption, distribution
and elimination. P-gp is highly expressed in several organs known to be important for drug
disposition. The localization of P-gp has been shown on the apical membrane of columnar
epithelial cells of intestine and proximal tubules in the kidney. In the liver P-gp is localized to
the canalicular side of the hepatocytes. The orientation of P-gp makes this transporter protein
important to protect the body against possible toxic response by excreting compounds into the
intestinal lumen, bile and urine. Inhibition of the P-gp transporter may also be the basis for
drug-drug interactions. The classical example is the increase in oral biovailability of digoxin
when co-administered with the P-gp inhibitor quinidine (Drescher et al., 2003; Igel et al.,
2007).
Several in vitro models have been used to investigate transporters and their role in
drug uptake and efflux in cells, revealing mechanistic understanding of drug transporter
affinities and kinetics. However, the results from in vitro systems have been difficult to fully
exploit in in vitro – in vivo scaling exercises. A key factor missing for useful in vitro-to-in
vivo translation of transporter data is information on the concentrations of the transporter
protein both in the in vitro system and in the organs handling the drug. Thus, a method
enabling the specific quantitative measurement of the membrane transporters concentration
would facilitate prediction of the relevance of individual transporters on human in vivo
pharmacokinetics of drugs and drug candidates when using in vitro model systems.
Traditionally, the enzyme-linked immunosorbent assay (ELISA) has been the
predominant method used for targeted quantification of a protein, providing good sensitivity
and throughput. However, the lack of antibodies with high specificity limits the use of
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immunological techniques. Moreover, the development of a high quality ELISA assay
requires a significant investment in time and resources. Alternative methods for targeted
protein quantification using mass spectrometry-based strategies have been developed to
address these issues. The core mass spectrometric technology that has emerged for protein
quantification is based on the concept of stable isotope dilution combined with multiple
reaction monitoring (MRM) (Gerber et al., 2003; Anderson et al., 2004; Carr and Anderson,
2008). The use of MRM mass spectrometry is based on the measurement of a “proteotypic”
tryptic peptide(s) that uniquely and stoichiometrically represents the protein target of interest.
Hence, a synthetic stable isotope labelled version of proteotypic peptide is used as an internal
standard, enabling protein concentration to be measured by comparing the signals from
isotopic labeled standard peptide to the endogenous peptide in the sample. During the last
years, liquid chromatography (LC) coupled to MRM mass spectrometry has been widely used
for protein quantification in biological and clinical samples (Pan et al., 2009). More recently,
the quantification of specific membrane transporters has been reported (Li et al., 2008, Li et
al., 2009, Li et al. 2009b, Kamiie et al., 2008). Li et al., 2008 demonstrated that the sensitivity
of an LC-MRM method for quantification of multidrug resistance associated protein 2
(MRP2) exceeded the sensitivity of an immunoblotting-assay. In another investigation, Li et
al. determined the absolute differences for breast cancer resistance protein (BCRP) and bile
salt export pump (BSEP) in livers and isolated hepatocytes across species. Li et al. (2009)
have also found that the protein levels of the hepatobiliary transporter MRP2 were
significantly different between species in freshly isolated and cryopreserved hepatocytes and
snap-frozen liver tissues from human, rat, monkey, and dog. Kamiie et al., 2008 developed an
LC-MRM method for simultaneous quantification of 36 membrane proteins (including some
transporter proteins) in brain capillary endothelial cells, liver and kidney of the mouse and
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Kawakami et al. (2011) reported the simultaneous determination of 11 human cytochrome
P450 enzymes with LC-MRM.
Here, we describe the development of a sensitive LC-MRM method for quantifying
human P-gp. The P-gp levels were measured in both HEK293 membrane vesicles expressing
human P-gp (Karlsson et al., 2010) and Caco-2 cells of different time in culture. Furthermore,
the P-gp protein expression level was correlated with mRNA expression level and functional
analysis. The generation of such quantitative data of transporters together with functional data
will be a key tool for the translation of results from various in vitro model systems to in vivo.
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Methods
Chemicals and reagents
Acetonitrile and water (LC-MS grade) were purchased from Fisher Scientific (Leicestershire,
UK). Phosphate buffered saline (PBS) pH 7.4, Colloidal Blue Staining kit, NuPAGE 4-12%
Bis-Tris gel, NuPAGE CDS sample buffer 4x, NuPAGE MES SDS running buffer 20x,
NuPAGE reducing agent, NuPAGE Antioxidant, and HBSS solution were all purchased from
Invitrogen (San Diego, CA, USA). Sodium carbonate (NaHCO3), disodium carbonate
(Na2CO3) and formic acid were obtained from Fluka (Steinheim, Germany). Precision plus
protein™ standards (Kaleidoscope), was obtained from Bio-Rad laboratories (Hercules, CA,
USA). Bovine serum albumin (BSA), ammonium bicarbonate (AMBIC), HEPES,
iodoacetamide (IAA), and TRIS-HCl were purchased from Sigma-Aldrich (Steinheim,
Germany). The protease inhibitor cocktail (complete™ Mini) was obtained from Roche
Applied Science (Mannheim, Germany), dithiothreitol (DTT) was obtained from Genomic
Solutions Inc. (Ann Arbor, MI, USA) and the PPS Silent Surfactant was obtained from
Protein Discovery Inc. (Knoxville, TN, USA). Sequencing grade-modified trypsin was a
product of Promega (Madison, WI, USA). The synthetic proteotypic peptide and its
corresponding stable-isotope-labelled (SIL) variant were purchased from Thermo Scientific
(Ulm, Germany). Tritiated N-methylquinidine and digoxin were purchased from RC Tritec
Ltd. (Teufen, Switzerland) and PerkinElmer Life and Analytical Sciences (Waltham, MA,
USA) respectively. All other chemicals were of at least analytical grade and obtained from
commercial sources.
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HEK-MDR1 vesicles preparation and functional assay
HEK293-EBNA cells were cultured and transiently transfected with human P-gp as described
earlier (Karlsson et al., 2010). Purified P-gp membrane vesicles from transfected HEK293-
EBNA cells were prepared as described previously (Karlsson et al., 2010). Membrane protein
content was determined using a BCA Protein Assay Kit (Pierce).
The vesicular transport activity was measured using a rapid filtration technique on 96 well
filter plates (Millipore MultiScreen HTS-FB plate). After a pre-incubation period of 5 min,
membrane vesicles (50 μg protein/75 μl reaction volume) were incubated at 37°C in the
presence or absence of 4 mM ATP in assay buffer (250 mM sucrose, 10 mM MgCl2, 10 mM
TRIS-HCl, pH 7.0) containing radiolabeled probe substrate [3H]-N-methyl-quinidine (NMQ,
1 μM, 3 μCi/ml) for 2 minutes. The uptake reaction was stopped by adding cold washing
buffer, immediately transferring the vesicles to the filter plate, and washing the filter with
washing buffer (250 mM sucrose, 100 mM NaCl, 10 mM TRIS-HCl, pH 7.0). The
radioactivity in the membrane vesicles retained on the filter was measured with a TopCount
scintillation counter (TopCount NXT, Packard Instrument, Meriden, CT, USA). ATP-
dependent transport (pmol/min/mg protein) was calculated as the difference between the
values obtained in the absence of ATP from those in the presence of ATP. Assays were run at
least in triplicates.
Caco-2 cell monolayer culture and transport assay
Caco-2 cells were purchased from ATCC (Rockville, MD, USA) at passage 18 and
maintained in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% heat
inactivated foetal calf bovine serum, 1% non-essential amino acids and 1.5% L-glutamine, in
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an atmosphere of 95% air and 5% CO2 at 37°C. All tissue culture media were obtained from
GIBCO, Life Technologies (Paisley, Scotland). For functional studies and P-gp quantitation
monolayer cultures were grown on polycarbonate culture inserts in medium also containing
antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). The cells were seeded at an initial
density of 2.5 x 105 cells per 1.13 cm2 filter (pore size 0.4 μm, Transwell®, Cat. No. 3401,
Corning Costar Corporation, Cambridge, MA, USA). The medium was changed every second
day. Cells for protein quantification were harvested by trypsinisation after 10 and 29 days in
culture on filters and pelleted by centrifugation at 300 g for 5 min.
At both culturing times (10 and 29 days), transport experiments were carried out in HBSS
buffered with 25 mM HEPES at pH 7.4. The Papp value of the P-gp substrate 3H-digoxin
(28nM) was determined in the apical to basolateral (A-B) and basolateral to apical (B-A)
direction in the absence and presence of verapamil (100 μM), a well-characterised inhibitor of
P-gp. The apparent permeability values (Papp) were calculated in all experiments according to
the equation Papp = (dQ/dt)/(A•C0) where dQ/dt = slope of the cumulative amount transported
during the time course of the period studied, A = monolayer culture area, and C0 = starting
concentration.
Functionality of P-gp protein was expressed as bidirectional transport ratio of the specific
probe substrate digoxin, and expressed as
Efflux ratio = Papp BA / Papp AB.
Inhibition of digoxin transport by verapamil in the Caco-2 cell system is interpreted as
functional evidence of P-gp mediated transport.
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mRNA isolation and quantification (RT-PCR)
mRNA of 6 filters with Caco-2 cells was extracted on day 10 and day 29 respectively, using a
phenol-chloroform extraction method (RNA-Stat) as described previously (Seithel et al.,
2006). mRNA quality was confirmed with integrity of 18S and 28S RNA bands in agarose gel
electrophoresis and RNA quantification was carried out with a Nanodrop spectrophotometer.
cDNA was prepared according to the manufacturer’s protocol using the Invitrogen
SuperScript II kit. RT-PCR was carried out using the Applied Biosystems assay-on-demand
for ABCB1 (Hs00184500_m1) and PPIA (Hs99999904_m1), on a 7500 instrument and
analysed using SDS2.3 software.
mRNA expression of ABCB1 was calculated using the deltaCT method against PPIA as a
reference gene.
delta CT = CT PPIA – CT ABCB1
Relative gene expression (REL) is then calculated as:
REL = 2-delta CT.
Extraction of membrane fraction
An overview over the extraction procedure is depicted in figure 1. The cell pellets were re-
suspended in 10 volumes of ice-cold 10 mM NaHCO3 pH 8 followed by adding protease
inhibitor cocktail according to the instructions of the manufacturer (Roche). The cells were
allowed to swell for 10 minutes and subjected to lysis using a glass-Dounce homogenizer
(25 strokes) on ice. Nuclei, unbroken cells and mitochondria were spun down (10000 g,
10 min, 4ºC). The post-nuclear supernatant, containing the membranes of interest, was
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adjusted to 100 mM Na2CO3 and sonicated for 20 min in order to disrupt electrostatic
interactions between the membranes and the membrane associated proteins. After the
carbonate-wash the membrane material was collected by ultracentrifugation (120.000 g, 1.5 h,
4ºC). The membrane pellet was resuspended in 50 mM AMBIC, using tip-sonication to
properly disperse the sample. The membrane samples were stored at -80ºC until future
analysis. Total protein concentrations of the various membrane fractions were determined
with the DC protein assay kit (Bio-Rad).
SDS-PAGE separation
The vesicle preparation of the HEK293 cells over-expressing human P-gp was diluted 10
times, 40 μg total protein loaded onto gel, and separated under reducing conditions on a
precast Bis-Tris SDS-PAGE gel. The protein bands were visualised by using the Colloidal
Blue Staining kit. The protein band corresponding to P-gp was cut out and dissected into 2
mm pieces that were transferred to a 1.5 mL Eppendorf microtube and subjected to in-gel
digestion. Briefly, the gel pieces were destained using 25 mM AMBIC in 70% acetonitrile
and dried by vacuum centrifugation for 10 min, reduced with 10 mM DTT at 56 ºC for 45 min
and subsequently IAA-alkylated for 30 min. Finally, the gel pieces were dried by vacuum
centrifugation for 15 min and then incubated with trypsin (0.15 μg) at 37°C overnight. The
tryptic digested peptides in the gel pieces were extracted with a solution containing 1% formic
acid in 5% acetonitrile in water. The peptide extract was stored at -80 ºC before analysis by
nano LC-chip-ESI-QTOF MS for proteotypic peptide identification.
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Identification of the proteotypic peptide
A 1 µL aliquot of the tryptic peptide extract was injected onto an LC/MS system consisting of
a 1200 Series system, LC-Chip Cube MS interface, and a 6520 ESI-QTOF mass spectrometer
(all Agilent Technologies). Chromatography was performed on an LC-Chip (Agilent
Technologies) that incorporated a 40 nL enrichment column and a 150 mm x 75 µm analytical
column packed with ZORBAX 300SB-C18, 5 µm particles. The tryptic peptides were loaded
onto the enrichment column with 97 % solvent A (2.5 % acetonitrile, 0.1 % formic acid) and
3 % B (95 % acetonitrile, 0.1 % formic acid ) at a flow rate of 4 µL/min. Elution was carried
out using an increasing gradient from 3 % B to 40 % B in 50 min, at a flow rate of
300 nL/min. The following Q-TOF conditions were used: drying gas 5 L/min (350°C);
fragmentor, 175V; skimmer, 60 V; capillary voltage, 1800 V; acquisition rate and time, 4
spectra/s (threshold 200 Abs, 0.01% rel.) and 250 ms/spectrum; MS scan range and rate, 296-
2500 at 4 Hz; MS/MS acquisition rate and time, 3 spectra/s (threshold 5 Abs, 0.01% rel.) and
333.3 ms/spectrum; MS/MS scan range and rate, 50-2500 at 3 Hz; collision energy slope, 3.3
V; offset, 2.5 V; auto MS/MS, 5 precursor; active exclusion on with 2 repeat and with release
after 0.1 min; preferred charge state, 2, 3, >3, unknown; internal reference mass correction
was enabled. The raw data files were exported to a Mascot Generic Format by MassHunter
(Version B.02.00) to create peak lists on the basis of the recorded fragmentation spectra.
Peptides and proteins were identified by Mascot V2.2.0 (Matrix Science, London, UK)
against the SwissProt database with a precursor mass tolerance of 10 ppm, a fragment ion
mass tolerance of ± 0.2 Da and trypsin specificity allowing for up to one missed cleavage.
Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation
was allowed as a variable modification. Peptides were rejected if the Mascot score was less
than the 95% confidence limit, based on the identity score of each peptide.
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Tryptic digestion protocol and preparation of calibration curve
An aliquot of 40 μl of each membrane sample was diluted 1:1 with PPS stock solution (0.2%
PPS, 10% acetonitrile in 50 mM AMBIC) in a 1.5 mL Eppendorf tube. The PPS enhanced the
extraction and solubilisation of hydrophobic membrane proteins and improved the in-solution
tryptic digestion of membrane proteins. The membrane samples were then reduced (10 mM
DTT at 50 ºC for 30 min) and alkylated (15 mM IAA in dark at room temperature for 45
min). After addition of internal standard, 25 fmol of stable isotope labelled (SIL) P-gp
proteotypic peptide (AGAVAEEV[13C615N1]LAAIR) the samples (2-50 µg protein) were
digested by trypsin in a final volume of 100 μL at 37 ºC for 6 h. The ratio of trypsin and
protein was 1 to 20. To avoid detergent interference in the subsequent mass spectrometric
analysis, the PPS was hydrolysed by adding 2.5 μL of neat formic acid for 1h. The samples
were centrifuged at 16.000 g for 10 min prior analysis by LC-ESI-MSMS. The external
calibration curve was prepared by spiking the synthetic proteotypic peptide
AGAVAEEVLAAIR in the digestion matrix with the addition of a fixed amount at 2500 pM
of the SIL internal standard peptide. Data were processed by integrating the appropriate peak
areas generated from the extracted ion chromatograms for the 13-mer analyte peptide and the
SIL internal standard peptide by MassHunter version B.01.04 (Agilent Technologies, Santa
Clara, CA, USA). The ratio of the peak area of the proteotypic peptide to the SIL peptide was
plotted against the concentration of the synthetic native peptide for constructing the regression
analysis.
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LC-MS/MS Quantification of P-gp
The selected proteotypic peptide was subjected to positive ion LC-MRM analysis utilizing an
LC/MS system comprising a UHPLC system (1290 Infinity binary pump, 1290 Infinity
autosampler with thermostat and 1290 Infinity thermostated column compartment, all Agilent
Technologies, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer (6460;
Agilent Technologies). Chromatography was performed on a 1.0 x 50 mm C18 column
(Acquity UPLC, BEH, C18, 1.7 μm size beads, 130 Å pore size, Waters). The mobile phase A
consisted of 2.5% acetonitrile, 0.1% formic acid and mobile phase B 95% acetonitrile, 0.1%
formic acid. The tryptic peptides were separated and eluted using a linear gradient starting
from 5% B that progressed to 60% B in 10 min. A sample volume of 10 μL was injected onto
the column at a flow rate of 0.2 mL/min. The MRM transitions for the proteotypic peptide
monitored represented the double-charged precursor ion (AGAVAEEVLAAIR)2H+ (m/z
635.4) to the single-charge product-ions y9, y8, and y7 with m/z 971.5, 900.5, and 771.5,
respectively. Similarly, the MRM transitions for the SIL internal standard peptide were the
corresponding double-charged precursor ion with m/z 638.9 to the single-charge product-ions
y9, y8, and y7 with m/z 978.5, 907.5, and 778.5, respectively. The instrument settings of the
6460 triple quadrupole mass spectrometer were as follows: drying gas temperature, 300 ºC;
drying gas flow, 6 L/min; nebuliser pressure, 40 psi; capillary voltage, 4500 V, sheath gas
temperature 350 ºC; sheath gas flow, 11 L/min, fragmentor voltage, 150 V; dwell time, 50ms;
collision energy for y9, y8, and y7-ions were 22, 18, and 18 eV, respectively.
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Data analysis
The presented functional data in HEK-P-gp vesicles was acquired of a minimum of two
experiments performed on different days. The comparison of P-gp protein amount between
different vesicle preparations and 10 and 29 days old Caco-2 cultures was statistically
analysed using Student’s t-test. A value p < 0.05 was regarded as statistically significant.
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Results
Selection of proteotypic peptide
The membrane protein preparation was separated by SDS-PAGE followed by Coomassie blue
staining. The P-gp protein band (141 kDa) was excised for in-gel tryptic digestion and the
extracted tryptic peptides were analysed by a nanoLC-ESI-QTOF mass spectrometer. The
acquired MS/MS data was then subjected to data base searching using the Mascot search
engine. Based on the intensity of the fragment ions and reproducibility, the 13-mer peptide
“AGAVAEEVLAAIR” which was produced by tryptic digestion of human P-gp protein was
selected as the proteotypic peptide with a Mascot score of 109. The MS spectrum and the
MS/MS spectrum of the selected proteotypic peptide, represented by
H-AGAVAEEVLAAIR-OH, are illustrated in Figure 2. The predominant fragment ions
(C-terminal y-ions) are corroborated with the sequence of the tryptic fragment.
The P-gp is a member of the superfamily of ATP-binding cassette transporters (ABC-
transporters), which is one of the largest families of transmembrane proteins. More
specifically, P-gp belongs to the ABC transporter subfamily B, which is composed of several
protein members as outlined in Table 1. A BLAST search was performed in order to ensure
that no exact amino acid sequence matches exist from any other ABC family member (table
1). The sequence alignment demonstrated that the AGAVAEEVLAAIR peptide is conserved
in P-gp across species of human, rat, mouse and dog. However, the selected proteotypic
peptide could easily be distinguished from the human ABCB-subfamily transporters listed in
Table 1. Therefore, the peptide sequence analysis based on the LC-MS/MS data along with
the confidence of a selective tryptic fragment for P-gp, this 13-mer tryptic peptide
(AGAVAEEVLAAIR) was selected as the surrogate marker for P-gp protein that can be used
for the development of an LC-MRM method.
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LC-MRM method development for quantification of P-gp
A synthetic variant of the selected proteotypic peptide (AGAVAEEVLAAIR) was purchased
along with its stable isotope labeled (SIL) variant (AGAVAEEVL*AAIR). The SIL peptide,
labeled (13C6, 15N1) at position of leucine, was used as an internal standard to normalize the
acquired data. The product ion spectra of P-gp and the corresponding SIL peptide are shown
in Figure 3. Three of the most intense transition ions y7, y8, y9 and y’7, y’8, y’9 were selected,
respectively, as the unique MRM signature for the development of a specific LC-MRM
method for P-gp quantification. The peak areas of all monitored parent to product ion
transitions of the synthetic proteotypic peptide were normalized by the peak area of the
corresponding MRM transitions of the SIL internal standard. The analytical procedure
included a standard curve placed at the beginning and end of the run to bracket the unknowns,
which resulted in a total of two measurements for each standard calibration level. The
calibration curve was linear over the concentration range of 10-5000 pM for P-gp and is
illustrated in Figure 4. The correlation coefficient (r2) was greater than 0.99. The limit of
quantification (LOQ) for P-gp was determined at 10 pM, as defined by a signal-to-noise ratio
response of 3 and with accuracy within ±10% and precision better than 10%. The response of
a blank sample analysed immediately following the analysis of a sample at the upper limit of
quantification was less than 1% of the response of the LOQ.
Improvement of detection sensitivity by minimizing analyte adsorption
A significant improvement in the detection sensitivity can be obtained for specific tryptic
peptides by a simple addition of 10% acetonitrile in the digestion matrix. Depending of the
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physicochemical properties a peptide can be more or less amenable for adsorption to plastic
surfaces (pipette tips, sample vials, tubing etc.). One option to avoid such binding would be
using siliconised or other low-binding surfaces that may be readily obtainable for tips and
vials, less straightforward for tubings and capillaries in the LC system. Another generic
approach is adding a small percentage of organic solvent to the buffer to prevent binding to
surfaces. It has previously been demonstrated that tryptic digestion in mixed organic (e.g.
acetonitrile)-aqueous solvent systems is highly efficient in terms of both rate of digestion and
amino acid sequence coverage (Russel et al., 2001). Russel et al. used as much as 80%
acetonitrile during trypsination. In this study we were able to obtain reproducible P-gp
quantifications when performing the digest with and without the presence of 10% acetonitrile.
In the case of the AGAVAEEVLAAIR peptide we observed almost a factor of 10 in
improvement of the detection sensitivity as illustrated in Figure 5, indicating that this peptide
has a tendency to adsorb which can be minimized by the addition of a small amount of an
organic modifier.
Validation of LC-MRM quantification for P-gp
To ensure appropriate performance of the established LC-MRM method, quality control (QC)
samples were prepared by spiking the synthetic proteotypic peptide in the membrane fraction
of a membrane vesicle preparation originating from HEK-293 cells at concentrations of 100
and 1000 pM subsequently followed by tryptic digestion overnight according to the protocol
previously described in Material and Methods. The SIL peptide was also spiked into each
sample as an internal standard prior trypsin digestion. The amount of QC peptide was
determined by subtracting the baseline of P-gp protein in the biological matrix. The
percentage relative error (RE %) of the prepared validation samples (100 and 1000 pM) at
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three independent days demonstrated the accuracy while the coefficient of variation (CV %)
represented the precision of the LC-MRM method. The validation results of the synthetic
proteotypic peptide are shown in Table 2. Reproducibility of the tryptic digestion of the
membrane preparation was evaluated by three independent digestions and LC-MRM
measurements of the P-gp concentration and resulted in a CV of less than 5% (data not
shown), clearly indicating that the trypsination process was reproducible.
P-gp mediated NMQ uptake in 2 batches of HEK-MDR1 vesicles
The ATP-dependent uptake of the P-gp substrate NMQ was determined in two different
vesicle batches, no 3 and no 6. The protein concentration of both preparations were very
similar (13 respective 10mg/mL), however the activity measurements differed significantly,
61 ± 11) pmol/min/mg protein and a +ATP/-ATP ratio of 4.4 in batch 3, whereas in batch 6
the active uptake was 220 ± 30) pmol/min/mg protein and the +ATP/-ATP ratio 9.3 (Table 3).
P-gp expression and transporter functionality in Caco-2 cell monolayers grown for 10 and 29
days
In accordance with previous publications, P-gp mRNA levels increase in Caco-.2 cell
monolayers with time in culture. In this study, a 3.4-fold increase in normalized mRNA levels
was observed between day 10 and day 29 (data not shown).
The bidirectional transport of the probe substrate digoxin was clearly direction dependent,
with more than 4.2-fold higher BA-transport on day 10 that increased to more than 10-fold on
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day 29. The calculated efflux ratio increased 2.2-fold between day 10 and day 29 (Table 3).
The efflux was inhibitable in presence of 100 µM verapamil (data not shown).
LC-MRM quantification of P-gp in HEK-MDR1 vesicles and Caco-2 cell monolayers grown
for 10 and 29 days
The LC-MRM method developed above was applied for the determination of P-gp protein
levels in the membrane fraction extracted from two HEK293-MDR1 vesicle batches and
Caco-2 cells that were cultured for 10 and 29 days, respectively. The specific MRM
transitions based on the chromatographic retention time and MS spectrum were used to detect
and quantify the endogenous P-gp proteotypic peptide by LC-MS/MS in MRM mode. Figure
8 shows the reconstituted ion chromatograms at different lengths of culture period of the
tryptic proteotypic P-gp fragment (AGAVAEEVLAAIR) released from trypsination of Caco-
2 cells. The human P-gp protein was determined at 4.0 fmol/µg protein (10 days) and
7.9 fmol/µg protein (29 days). The amount of P-gp in the cell sample grown for 29 days was
about 2-fold higher than in the sample that was grown for 10 days. HEK-MDR1 vesicle
batches differed nearly 4-fold in their P-gp protein contents, 8.2 and 32.0 fmol/µg protein in
batch 3 and batch 6 respectively.
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Discussion
P-gp is abundant in all organs important for the disposition of many drugs in the body (Lin
and Yamazaki, 2003; Lee et al., 2010; Sun et al., 2004). The combination of the wide
distribution in the body and the broad substrate spectrum for P-gp implies that mechanistic
and quantitative models are needed to understand how this transporter impacts
pharmacokinetics of drugs in the body. Caco-2 cells, MDCK-MDR1 and human P-gp
transfected vesicles are currently common in-vitro systems used to address the mechanistic
relevance of substances as substrates or inhibitors of P-gp (FDA draft guidance DDI, 2006).
In particular interpretation of inhibition data from in vitro models are increasingly well
understood and are in many cases successfully translated to relevant information for the in
vivo situation (Fenner et al., 2009, Cook et al. 2010). In contrast, extrapolation of substrate
kinetic data from in vitro models to in vivo pharmacokinetics remains more challenging. Even
though P-gp substrates can with good confidence be identified using in-vitro systems the
quantitative relevance of the active transport process for the pharmacokinetics in vivo is still
difficult to predict from in-vitro data.
In comparison, successful in-vitro in-vivo extrapolation for hepatic metabolic clearance has
been applied for CYP mediated metabolism. A key factor for the well accepted in vivo
prediction of hepatic clearance is the possibility to measure the specific CYP content in the
liver tissue and in the various in vitro systems used. The amount of active protein in the in
vitro system can thus be directly contrasted and related to the amount of active protein in the
tissue (Barter et al., 2007). Today, similar scaling factors and extrapolation equations for
membrane transporters are scarcely available. Transporter substrate kinetics can be compared
and ranked in affinities in the same in-vitro system. But for a useful comparison between
different in vitro systems and prediction to in vivo, the absolute amount of functional protein
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is needed. The protein atlas project (Nilsson et al., 2005) is to some extent filling this gap with
semi-quantitative information from protein data (Western blot and IHC) and mRNA
expression. However it is highly warranted to be able to use specific scaling factors that
describe the amount of transporter protein per cell unit or gram tissue. In a recent publication
Bolger et al. (2009) applied relative expression scores, normalised to ileum to account for
varying P-gp levels in different intestinal regions and simulated PK- and absorption profiles
after different doses using an advanced compartmental absorption and transit (ACAT) model
in GastroPlus simulations. The relative expression profiles from literature sources were
empirically compared in the simulation model, and built into physiological characteristics of
the small and large intestines for the simulation. The authors proposed an ACAT model that
was able to accurately account for the regional and temporal changes in concentration and
carrier-mediated transport and reproduced the nonlinear dose dependence observed in vivo.
In the present study we report the successful development of a LC-MRM method to quantify
the transport protein P-gp (ABCB1) in in vitro systems often used for
P-gp studies and for the first time relate protein abundance to transporter functionality in these
systems. Several critical factors that affect both the sensitivity and specificity of an assay have
to be considered in the selection of a proteotypic peptide for development of quantitative
protein assays based on a proteolytic peptide surrogate marker (the proteotypic peptide).
Firstly, the peptide amino acid sequence has to be unique to the target protein and contain no
missed cleavage sites. Secondly, the peptide must be reproducibly observed in proteolytic
digests. Thirdly, the amino acid sequence of the peptide should not contain amino acids that
are susceptible to chemical modifications. The tryptic fragment used in this study as marker
peptide, is highly specific among proteins from the ABCB-transporter subfamily. In contrast
to the method published recently by Zhang et al (2011), where mouse mdr1b was not detected
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using a 9‐mer peptide as selective tryptic fragment for P‐gp, the present method will quantify
all P‐gp protein in human and preclinical species, including the rodent forms 1a and 1b.
ABCB1 is detected selectively from proteins ABCB4 and ABCB11, which have the closest
related protein sequences with 2 and 6 amino acids difference respectively. Other members of
the ABCB family are not showing a homologous sequence. Since protein quantification by
LC-MRM methodology is based on the measurement of a tryptic peptide, it is critical that the
tryptic digestion should achieve 100% efficiency in order to accurately quantify the protein of
interest in a biological sample. The transmembrane domain should be avoided when selecting
a proteotypic peptide. Typically, trypsin has limited availability in the transmembrane domain
that is situated within the lipid bilayer and hereby “protected”. Moreover, even if trypsination
occurs in the transmembrane domain it may be difficult to extract the peptides from the lipid
bilayer. The proteotypic peptide identified for P-gp in this study, is not located within a
transmemebrane domain. In our experience the selection of proteotypic peptide is difficult to
predict by in-silico experiments and until more reliable algorithms have been developed we
prefer to conduct wet lab trypsinations subsequently followed by LC-MS experiments.
Ideally, a pure protein is used as reference standard to confirm the complete digestion of the
target protein. However, in the present study the method was developed without a pure
protein standard due to the lack of commercial availability of purified P-gp. This is not an
uncommon situation for quantification of membrane proteins. One way of addressing this
issue is to synthesize a slightly longer surrogate digestion substrate peptide that contains the
amino acid residues of the proteotypic peptide (Li et al. 2008). However, the structure of a
large and complex transmembrane protein is significantly different to digest as compared to a
surrogate digestion substrate peptide. The use of gene-transfected cells overexpressing P-gp
enabled us to conduct tryptic mapping and subsequent identification of a suitable proteotypic
peptide. Precision and accuracy of the LC-MRM method are within 10% and meet quality
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standards for bioanalytical methods. The method produced reproducible quantification data
from different in-vitro matrices on different occasions. The relevance of the membrane
transporter abundance data is confirmed by a good correlation to transporter functionality in
the two different in-vitro systems. The amount of P-gp in the Caco-2 cell samples from cells
grown for 29 days was 2-fold higher than in cells grown for 10 days, in very good agreement
with the functional data on digoxin efflux, which increased 2.2-fold between the two cell
cultivating times. Similar concordance between NMQ-uptake functionality in inside-out
HEK-MDR1 vesicles and protein contents was observed. A 3.6-fold increase in functional
activity was reflected by a 3.9-fold increase in protein content. The observed abundance –
functionality relationships in both membrane vesicle preparations and monolayer grown cells
show that the developed LC-MRM method is applicable to quantify the specific protein
contents in the in-vitro systems.
The presented LCMS method to quantify membrane protein levels enables accurate
determinations of human P-gp protein abundance in isolated cells. The high analytical
sensitivity of the method is promising for its application to different types of human tissues in
the future.
Based on the fact that the proteotypic peptide used in this study is a homologous sequence in
human, rat, dog, mouse P-gp, the technology is readily transferable to quantitation of non-
human P-gp and will enable direct interspecies comparison of protein abundance. In addition,
comparison of protein levels between species and tissues will be possible. Such knowledge is
warranted to guide interpretation of observed inter-species differences in pharmacokinetic
profiles. Additionally, also inter-individual differences in P-gp protein levels between donors
can be quantified and ameliorate understanding of human variability.
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In summary, the presented method to quantify P-gp by LC-MRM will facilitate future
methodologies to bridge between expression systems and cell/tissue models and to scale from
in-vitro models to whole organs. Availability of such scaling factors will be instrumental to
successful extrapolation to human, to understand in a quantitative manner the impact of P-gp
mediated transport on overall pharmacokinetic profiles and drug distribution.
Since the P-gp quantitative method developed in the present study also could be used in
animals the application could be extended to describe levels and variations of P-gp levels in
various organs in animals and how this may affect pharmacokinetics in vivo.
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Authorship Contributions
Participated in research design: Miliotis, Palm, Andersson, and Hilgendorf.
Conducted experiments: Miliotis, Ali, and Hilgendorf.
Contributed new reagents or analytic tools: Miliotis, Lundquist, and Ahnoff.
Performed data analysis: Miliotis, Ali, Palm, and Hilgendorf.
Wrote or contributed to the writing of the manuscript: Miliotis, Palm, Andersson, and
Hilgendorf.
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Figure legends
Figure 1: Schematic overview membrane preparation procedure and identification of
proteotypic peptide (left) and sample workup for LCMS quantification of specific membrane
protein (right).
Figure 2: MS- and MS/MS-spectra of the proteotypic peptide for P-gp identified on a Q-TOF
instrument. (A) The MS spectrum of H-AGAVAEEVLAAIR-OH (double charge, m/z
635.36), the pre-cursor ion of the proteotypic peptide. The inserted magnified spectrum
depicts the monoisotopic resolution in the mass range 630 to 640. (B) Product ion mass
spectrum from the collision-induced dissociation of m/z 635.36 precursor ion
(AGAVAEEVLAAIR)2H+ to single charge products, predominant fragment ions (C-terminal
y-ions) are highlighted.
Figure 3: Upper panel depicts the y-ions formed from collision induced transition from the
proteotypic peptide, the three most intense transition ions, y-ions of m/z 971.5, 900.5, 771.5,
were selected to develop the LC-MRM method. In the lower panel the corresponding y-ions
from the heavy peptide (internal standard) are shown, for all a mass shift of +7 Da adds to the
corresponding transitions of the SIL peptide due to the isotope labeling.
Figure 4: Representative calibration curve over the concentration range 10 to 5000 pmol/L.
The concentration of SIL internal standard was fixed at 2500 pmol/L. Each calibration
standard was measured in duplicate before and after each analytical run to bracket the
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unknowns. The insert represents the extracted ion chromatogram at the lowest point in the
standard curve (10 pmol/L), showing a clear signal against baseline.
Figure 5: The detection sensitivity was significantly improved after addition of 10%
acetonitrile to the digestion matrix.
Figure 6: Representative reconstructed mass chromatograms of P-gp proteotypic peptide
(transition of m/z 635.4 --> m/z 971.5) from trypsinated membrane preparations of Caco-2
cells cultured for 10 days (upper panel) and 29 days (lower panel), respectively.
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Tables
Table 1: The selected proteotypic peptide, AGAVAEEVLAAIR, for human P-gp is unique
and distinct from other human transporters from the ABCB subfamily, different amino acids
are depicted in bold italics. The sequence is specific for an ABCB1 region that is highly
conserved between species, and found identical in abcb1a and 1b isoforms in mouse and rat,
and abcb1 in dog and human.
ABCB protein and species Sequence agreement proteotypic peptide
ABCB1_HUMAN AGAVAEEVLAAIR
ABCB1a_RAT AGAVAEEVLAAIR
ABCB1b_RAT AGAVAEEVLAAIR
ABCB1a_MOUSE AGAVAEEVLAAIR
ABCB1b_MOUSE AGAVAEEVLAAIR
ABCB1_DOG AGAVAEEVLAAIR
ABCB4_HUMAN AGAVAEEALGAIR
ABCB11_HUMAN AGVVADEVISSMR
ABCB5_HUMAN N/A
ABCB2_HUMAN N/A
ABCB3_HUMAN N/A
ABCB9_HUMAN N/A
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ABCB8_HUMAN N/A
ABCB10_HUMAN N/A
ABCB6_HUMAN N/A
ABCB7_HUMAN N/A
N/A: no sequence homology to identified ABCB1 tryptic digestion fragment
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Table 2: Intra- and interday validation of P-gp synthetic proteotypic peptide quantification.
The synthetic proteotypic peptide was spiked to the membrane fraction of a membrane vesicle
preparation originating from untransfected HEK-293 cells (matrix) at two concentrations (100
and 1000 pmoles/L). Preparations were performed at three independent occasions.
Experiment
Matrix
concentration (pM)
100 1000
Day 1 n
Mean
Accuracy
(%RE)
Precision
(%CV)
50.8
3
91.2
-8.8
9.6
3
1017.6
1.8
10.9
Day 2 n
Mean
Accuracy
(%RE)
Precision
(%CV)
41.6
3
113.1
13.1
12.1
3
935.7
-6.4
8.5
Day 3 n
Mean
49.1
3
87.4
3
918.5
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Accuracy
(%RE)
Precision
(%CV)
-12.6
11.3
-8.1
10.2
Inter-day n
Mean
Accuracy
(%RE)
Precision
(%CV)
9
97.2
-2.8
12.0
9
957.3
-4.2
10.7
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Table 3. P-gp content as determined by LC-MRM-MS and functionality in two batches of
HEK-MDR1 vesicles and in 10 and 29-day old Caco-2 cell monolayers. P-gp activity is
determined as 3H-NMQ uptake in inside out HEK-MD1 vesicles and 3H-digoxin efflux ratio,
as described in the methods section; values are given mean ± SD, n=3-4.
HEK293 – MDR1 vesicles Caco-2 cell monolayers
3H-NMQ
uptake
(pmol/min/mg
protein)
P-gp content
(pmol P-gp /
mg total
protein)
3H-
digoxin
efflux
ratio
P-gp content
(pmol P-gp /
mg total
protein)
batch 3 61 ± 11 8.15 10 days 4.6 ± 0.1 4.00
batch 6 220 ± 30 32.0 29 days 10.2 ± 2.4 7.89
Fold
difference
(batch 6/
batch 3)
3.6 3.9
Fold
difference
(29 days/ 10
days)
2.2 2.0
This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on September 23, 2011 as DOI: 10.1124/dmd.111.040774
at ASPE
T Journals on A
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Cells
Lysis of cells (Dounce)
UCF 120.000g (1.5h)
Add protease inhibitor cocktail
Centrifugation 10.000g, wash
Tip-sonication
SDS-PAGE separationCoomassie Blue stain
Dissect
In-solution tryptic digestion
Identification of proteotypic peptideLC-MRM method development
LC MRM-MSQuantification
Vesicle preparation
Nano LC-ESIQ-TOF-MS
In-gel tryptic digestion
Figure 1
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D Fast Forw
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y9y8
y7y6
y5
y4
A
B
Figure 2
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atted. The final version m
ay differ from this version.
DM
D Fast Forw
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OI: 10.1124/dm
d.111.040774 at ASPET Journals on April 5, 2020 dmd.aspetjournals.org Downloaded from
Figure 3
x103
0
1
2
3
+ Product Ion (10.849 min) (635.36139[z=2] -> **) 081006_tmi_H+L_5fmol_SRM2.d 271.17 971.55
430.27
635.36
72.08
1070.62y10
y9
y8
y7
y6y5
y4
y3
y2y1
y7y8
y9
A-G-A-V--A--E--E-V-L(*)-A-A-I-R
x103
0
1
2
978.56271.16
550.37649.45
72.08
1077.62
Counts vs. Mass-to-Charge (m/z)100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 19002000
y’10
y’9
y’1y’2
y’3
y’4
y’5
y’6 y’7
y’8
+ Product Ion (10.862 min) (638.86139[z=2] -> **) 081006_tmi_H+L_5fmol_SRM2.d
This article has not been copyedited and form
atted. The final version m
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DM
D Fast Forw
ard. Published on September 23, 2011 as D
OI: 10.1124/dm
d.111.040774 at ASPET Journals on April 5, 2020 dmd.aspetjournals.org Downloaded from
Figure 4
Relative Respo
LOQ: 10 pM
m/z 635.4 � m/z 971.5
(10-10000) pM (r2 = 0.995)
2
3
1
4
0
1 32 4
Re
lative
Re
sp
on
se
Relative Concentration0
x101
4.5
5
5.5
6
+ESI MRM Frag=150.0V CID@** (635.4 -> 971.6) 100514_tmi_Pgp_10pM_run1.d 1
This article has not been copyedited and form
atted. The final version m
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DM
D Fast Forw
ard. Published on September 23, 2011 as D
OI: 10.1124/dm
d.111.040774 at ASPET Journals on April 5, 2020 dmd.aspetjournals.org Downloaded from
10% ACN
Without ACN
x103
1
0.5
Counts vs. Acquisition Time (min)3 43.5 4.5
Figure 5
This article has not been copyedited and form
atted. The final version m
ay differ from this version.
DM
D Fast Forw
ard. Published on September 23, 2011 as D
OI: 10.1124/dm
d.111.040774 at ASPET Journals on April 5, 2020 dmd.aspetjournals.org Downloaded from
x101
5
6
7
8
9
+ESI MRM Frag=150.0V CID@** (635.4 -> 971.6) 100514_tmi_Pgp10days
4.446
x102
0.5
1
1.5+ESI MRM Frag=150.0V CID@** (635.4 -> 971.6) 100514_tmi_Pgp29days
4.450
Counts vs. Acquisition Time (min)
4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5
Figure 6
This article has not been copyedited and form
atted. The final version m
ay differ from this version.
DM
D Fast Forw
ard. Published on September 23, 2011 as D
OI: 10.1124/dm
d.111.040774 at ASPET Journals on April 5, 2020 dmd.aspetjournals.org Downloaded from