Development of a highly sensitive method using LC-MRM to...

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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.

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

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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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References

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spectrometric quantitation of peptides and proteins using Stable Isotope Standards and

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Barter ZE, Bayliss MK, Beaune PH, Boobis AR, Carlile DJ, Edwards RJ, et al. (2007) Scaling

Factors for the Extrapolation of In Vivo Metabolic Drug Clearance From In Vitro Data:

Reaching a Consensus on Values of Human Microsomal Protein and Hepatocellularity Per

Gram of Liver. Curr Drug Metab. 8:33-45

Bolger M, Lukacova V, Woltosz W (2009) Simulations of the Nonlinear Dose Dependence

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Carr SA, Anderson L (2008) Protein Quantitation through Targeted Mass Spectrometry: The

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Drescher S, Glaeser H, Murdter T, Hitzl M, Eichelbaum M, Fromm MF (2003) P-

glycoprotein-mediated intestinal and biliary digoxin transport in humans. Clin Pharmacol

Ther. 73:223-231.

Cook JA, Feng B, Fenner KS, Kempshall S, Liu R, Rotter C, et al. (2010) Refining the In

Vitro and In Vivo Critical Parameters for P-Glycoprotein, [I]/IC50 and [I2]/IC50, That Allow


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for the Exclusion of Drug Candidates from Clinical Digoxin Interaction Studies. Molecular

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/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf

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Drug Interactions Mediated Through P-Glycoprotein: Clinical Relevance and In Vitro–In

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absorption of digoxin from the human jejunum due to inhibition of intestinal transporter-

mediated efflux. Clin Pharmacokinet. 46:777-785.

Kamiie J, Ohtsuki S, Iwase R, Ohmine, Katsukura Y, Yanai K, Sekine Y, Uchida Y, Ito S,

and Terasaki T (2008) Quantitative atlas of membrane transporter proteins: development and

application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-

silico peptide selection criteria. Pharm Res 25:1469-1483.

Karlsson JE, Heddle C, Rozkov A, Rotticci-Mulder J, Tuvesson O, Hilgendorf C, Andersson

TB (2010) High-Activity P-Glycoprotein, Multidrug Resistance Protein 2, and Breast Cancer


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Resistance Protein Membrane Vesicles Prepared from Transiently Transfected Human

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clinical importance and a consideration of disease states. Expert Opinion Drug Metab Toxicol.

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Li N, Nemirovskiy OV, Zhang Y, Yuan H, Mo J, Ji C, Zhang B, Brayman TG, Lepsy C,

Heath TG, and Lai Y (2008) Absolute quantification of multidrug resistance-associated

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quantification and comparison of bile salt export pump and breast cancer resistance protein in

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Li N, Zhang Y, Hua F, and Lai Y (2009) Absolute difference of hepatobiliary transporter

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Lin JH, Yamazaki M. (2003) Role of P-glycoprotein in pharmacokinetics: clinical

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(2009) Mass spectrometry based targeted protein quantification: methods and applications. J

Proteome Res 787:787-797.

Zhang Y, Li N, Brown PW, Ozer JS, and Lai Y. (2011) Liquid chromatography/tandem mass

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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


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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

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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y9y8

y7y6

y5

y4

A

B

Figure 2




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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




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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




DM

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10% ACN

Without ACN

x103

1

0.5

Counts vs. Acquisition Time (min)3 43.5 4.5

Figure 5




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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




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