homogenates through multiple reaction monitoring mass
spectrometry
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Une quantification sélective et sensible de la protéine du cytochrome P450 3A4 dans des homogénats de foie humain par spectrométrie de en mode d’acquisition « multiple reaction monitoring.
RÉSUMÉ
La présente étude visait à établir une méthode de spectrométrie de masse en mode d’acquisition « multiple reaction monitoring » (MRM-MS) pour quantifier l’enzyme cytochrome P450 (CYP) 3A4 dans des homogénats de foie humain. Des échantillons de foie ont été soumis à une digestion par la trypsine. L’analyse par MRM-MS a été réalisée à l’aide de 3 transitions optimisées sur 1 unique peptide synthétique purifié de CYP3A4, et normalisée à la calnexine. Les coefficients de variation pour la précision et la reproductibilité de la mesure par le MRM-MS ont également été déterminés. La méthode a été appliquée aux échantillons de foie de 10 donneurs non-cholestatiques et 34 patients cholestatiques avec cholangite biliaire primitive (n = 12; CBP), cholangite sclérosante primitive (n = 10; CSP) ou d'une maladie alcoolique du foie (n = 12; MAF). La méthode établie présente une sensibilité élevée avec une limite de détection inférieure à 5 fmol, et a été appliquée avec succès pour la quantification absolue et relative de l’enzyme CYP3A4 à la fois dans les fractions homogénéisées et microsomales de foie humains. Lorsque tous les groupes ont été analysées en même temps, une corrélation significative a été observée pour la quantification de la protéine CYP3A4 par la méthode MRM-MS dans des homogénats et des microsomes (r = 0,49, p <0,001). Aucune différence statistiquement significative n'a été détectée pour les niveaux de CYP3A4 dans la CSP, la CBP, la MAF et les échantillons contrôls. Enfin, la quantification par MRM-MS de CYP3A4 dans des homogénats corrèle également (r = 0,44; p <0,05) avec le niveau d'activité enzymatique provenant des mêmes échantillons, tel que déterminé par la mesure de la conversion de l’acide chénodéoxycholique en l’acide hyocholique.
Conclusion: La méthode établie fournit un outil sensible pour quantifier le cytochrome
P450 dans des homogénats de foie humain contrôle et ceux provenant de patients qui présentent des dommages hépatiques sévères/ou chroniques.
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SELECTIVE AND SENSITIVE QUANTIFICATION OF THE CYTOCHROME P450 3A4 PROTEIN IN HUMAN LIVER HOMOGENATES THROUGH MULTIPLE REACTION MONITORING MASS SPECTROMETRY.
Anna Cieślak1, Isabelle Kelly2, Jocelyn Trottier1, Mélanie Verreault1, Ewa Wunsch3, Piotr
Milkiewicz3,4, Guy Poirier2, Arnaud Droit2 and Olivier Barbier1
1 Laboratory of Molecular Pharmacology, CHU de Québec Research Centre and the
Faculty of Pharmacy, Laval University, Québec, Canada.
2 Proteomics Platform of the Québec Genomics Center, CHU de Québec Research Centre,
Québec, Canada.
3 Liver Research Laboratories, Pomeranian Medical University, Szczecin, Poland.
4 Liver and Internal Medicine Unit, Department of Transplant and Liver Surgery, Medical
University of Warsaw, Warsaw, Poland.
Corresponding author: Olivier Barbier Ph.D
Laboratory of Molecular Pharmacology, CHU de Québec Research Centre, 2705, boulevard Laurier,
Québec (QC) G1V 4G2, CANADA Phone: 418 654 2296
Fax: 418 654 2769
Email: olivier.barbier@crchul.ulaval.ca
ABBREVIATIONS: ALD, alcoholic liver disease; CDCA, chenodeoxycholic acid; CYP3A4,
cytochrome (CYP) P450 3A4; HCA, hyocholic acid; MRM-MS, multiple reaction monitoring mass spectrometry; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; SIS, stable isotope-standard.
KEY WORDS: Alcoholic liver disease; cytochrome P450 (CYP)3A4; MRM-MS quantification;
primary biliary cholangitis; primary sclerosing cholangitis.
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ABSTRACT
The present study aimed at establishing a sensitive multiple reaction monitoring- mass spectrometry (MRM-MS) method for the quantification of the drug metabolizing cytochrome P450 (CYP)3A4 enzyme in human liver homogenates. Liver samples were subjected to trypsin digestion. MRM-MS analyses were performed using 3 transitions optimized on 1 purified synthetic peptide unique to CYP3A4 and the standardizing protein, calnexin. Coefficient of variations for the precision and reproducibility of the MRM-MS measurement were also determined. The method was applied to liver samples from 10 non-cholestatic donors and 34 cholestatic patients with primary biliary cholangitis (n=12; PBC), primary sclerosing cholangitis (n=10; PSC) or alcoholic liver disease (n=12; ALD). The established method presented high sensitivity with limit of detection lower than 5fmol, and was successfully applied for the absolute and relative quantification of CYP3A4 in both whole liver homogenate and microsomal fractions. When all groups were analyzed together, a significant correlation was observed for the MRM-based CYP3A4 protein quantification in homogenates and microsomes (r=0.49, p<0.001). No statistically significant difference was detected between CYP3A4 levels in PSC, PBC, ALD and control samples. Finally, the MRM-MS quantification of CYP3A4 in homogenates also correlated (r=0.44; p<0.05) with the level of enzyme activity in the same samples, as determined by measuring the chenodeoxycholic to hyocholic acid conversion.
Conclusion: The established method provides a sensitive tool to evaluate cytochrome
P450 in human liver homogenates from patients with normal or chronic/severe hepatic injury.
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STATEMENT OF SIGNIFICANCE OF THE STUDY
Determination of the CYP3A4 protein content in human liver samples could help to evaluate treatment regimen in clinics. Multiple reaction monitoring-mass spectrometry (MRM- MS) methods with internal stable isotope-labeled standard peptides (SIS) have been successfully employed for cytochrome P450’s quantification in the microsomal fraction of human liver samples [1, 2]. However, microsome purification is a time-consuming sample preparation step [1, 3] and the use of heterogeneous microsome suspension as analytical matrixes can reduce the reproducibility of MRM-MS methods. The present study reports the first method allowing the sensitive, selective and reproducible determination of CYP3A4 protein levels in human liver homogenates.
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INTRODUCTION
The cytochrome P450 (CYP)3A4 enzyme is a member of the CYP superfamily of proteins, predominantly expressed in the liver and gastrointestinal tract where it participates in the metabolism of many drugs and xenobiotics [497]. In addition, CYP3A4 also catalyzes the oxidative metabolism of several endogenous compounds such as estrogens or bile acids (BAs) [497-499].
Due to its pivotal role in the biotransformation of active substrates, the variability in CYP3A4 expression and activity is considered to be a major determinant of the individual response to therapeutic treatment [48, 49]. Determination of the CYP3A4 protein content in human liver samples could therefore help to further evaluate treatment regimen in clinics. Biochemical approaches such as reverse transcription PCR (RT-PCR) [500, 501], differential gel electrophoresis (DIGE) [502] and Western blotting [503] have been developed for CYP3A4 quantification. However, these techniques suffer from numerous limitations (low correlation between mRNA and protein expression levels, low sensitivity, variability across instrumentation platforms, lack of specific antibodies), and only provide an estimation of the protein level [504]. Additional high-throughput, sensitive and selective methods are therefore required for CYP3A4 enzyme determination. Recently, mass spectrometry (MS)-based proteomic methods have become the cornerstone for protein quantification. The most commonly used techniques, namely Stable Isotope Labeling with Amino acids in Cell culture (SILAC), Isotope Coded Affinity Tags (ICAT) and isobaric Tags for Relative and Absolute Quantification (iTRAQ), are based on the identification of trypsin- digested proteotypic peptides unique to the protein of interest that can be used as quantification markers [505-507]. However, limitations of those methods with the regard to quantification accuracy and throughput are well established [508, 509]. The multiple reaction monitoring (MRM)-MS method with internal stable isotope-labeled standard peptides (SIS) emerged as an alternative, highly selective and sensitive approach for the absolute targeted protein determination in complex biological samples [510]. Such methods have been successfully employed for CYPs quantification in the microsomal fraction of human and murine liver samples [511-516]. However, microsomes purification is a time-consuming sample-preparation step [517, 518] and the use of heterogeneous microsomes suspension as analytical matrixes can reduce the reproducibility of MRM-MS quantification. To overcome such a problem, we have developed a MRM-MS-based CYP3A4 quantification method which can be used with both microsomes and
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homogenates from human tissue samples. After a thorough validation, the method has been successfully used to determine the CYP3A4 protein levels in clinically relevant human liver samples obtained from control volunteers, and cholestatic patients suffering from alcoholic liver disease (ALD), primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC). To the best of our knowledge, the present study is the first one reporting a sensitive MRM-MS assay allowing the sensitive and reproducible determination of the CYP3A4 protein in human liver homogenates.
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MATERIALS AND METHODS
Chemicals and reagents
Ammonium bicarbonate, sodium deoxycholate, iodoacetamide, dithiothreitol, K2HPO4, KH2PO4, glycerol, ethylenediaminetetraacetic acid (EDTA), formic acid,
trifluoroacetic acid (TFA), acetic acid, nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) and acetone were purchased from Sigma (all more than 97% pure; St. Louis, MO, USA). Tris-(2-carboxyethyl)phosphine was from Thermo Scientific (Rockford, Il, USA), while modified sequencing-grade trypsin was obtained from Promega (Madison, WI, USA) and protease inhibitors cocktail (pepstatin, leupeptin) from Sigma. Normal and deuterated chenodeoxycholic (CDCA) and hyodeoxycholic (HCA) acids were purchased from Steraloids Inc. (Newport, RI, USA) and C/D/N Isotopes Inc. (Pointe-Claire, Qc, Canada), respectively. Strata X and Synergie RP Hydro columns were from Phenomenex (Torrance, CA, USA). Purified synthetic peptides containing [13C
6,15N2]Lys and [13C6,15N4]Arg were obtained from Pierce (Rockford, IL, USA).
After synthesis, the peptides were purified by HPLC with subsequent quantification by amino acid analysis.
All mobile phases and solutions were prepared with LC/MS grade solvents (i.e. water, acetonitrile (ACN) and methanol), and were from VWR Canada (Mississauga, ON, Canada).
Non cholestatic, ALD, PBC and PSC donors
This study received IRB approval from clinical study review boards at the CHU de Québec research centre and Pomeranian Medical University. Informed consent was obtained from each volunteer.
Non-cholestatic liver samples were obtained as previously described [59, 519, 520] and correspond to liver tissues from kidney donors of Caucasian origin obtained at the time of kidney donation (Supplemental Table 1).
The study group consisted of 34 cirrhotic patients: 12 with PBC, 10 with PSC and 12 with ALD (Supplemental Table 1). Alcoholic etiology of a liver disease was considered in case of a reliable history of prolonged alcohol abuse and after exclusion of other causes of a liver damage. The diagnosis of PSC was confirmed by imaging techniques (magnetic resonance cholangiopancreatography-MRCP or endoscopic retrograde cholangiopancreatography-ERCP) and/or liver histology. The diagnosis of PBC was done
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according to the widely accepted criteria, which included: elevated alkaline phosphatase (ALP), typical liver histology and positive titers of antimitochondrial autoantibodies (AMA). Liver cirrhosis was confirmed with liver biopsy or typical appearance of the liver on abdominal ultrasound and/or computerized tomography scan. The samples (liver tissues) were collected from explanted livers during/shortly after liver transplantation and immediately frozen at -80°C. All patients with PBC and PSC, and none of the subjects with ALD were treated with ursodeoxycholic acid (URSO®) at the time of tissue collection. For MRM-MS quantification, 34 samples divided into 3 groups (PBC, PSC and ALD) and 10 samples (NC) for non-cholestatic controls were analyzed.
Protein extraction from liver homogenates
Frozen liver tissues were weighted (30-50 mg), and disrupted using a mortar and pestle. Samples were kept frozen on dry-ice and grinded to fine powder. An ice-cold lysis buffer (ammonium bicarbonate: 50 mM, pH 8; DTT: 50 mM; sodium deoxycholate: 0.5%; and a protease inhibitors cocktail) was added, prior to sonication on ice with a sonic dismembrator (Fisher; 1 sec. pulse for 20-times). Samples were centrifuged 10min at
16,000 g, and supernatants were precipitated with 5 volumes of acetone overnight at -
20°C. Precipitated proteins were centrifuged 15min. at 16,000 g, and protein pellets were then air-dried prior to being resuspended in ammonium bicarbonate (50 mM, pH8) containing 1% sodium deoxycholate. Finally, the protein concentration of each sample was determined by colorimetric Bradford assay (Bio-Rad Labs., Hercules, CA).
Isolation of Microsomal Proteins
Purification of microsomal fraction from liver samples was performed as previously reported [518]. Briefly, liver samples were homogenized in K2HPO4 (0.1 M, pH 7.4),
KH2PO4 (0.1 M, pH 7.4), glycerol (20%), EDTA (1 mM), DTT (1 mM) using a potter-glass-
col type homogenizer (Glas-Col LLC, Terre Haute, IN, USA) with a teflon pestle at 4°C. Homogenates were centrifuged at 12,000g for 20 min. at 4°C. Supernatants were then centrifuged at 105,000 g for 1 h at 4°C. Microsome pellets were resuspended in homogenization buffer at a concentration of 5μg/μL and stored at −80°C until trypsin digestion.
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Solubilization and in-solution digestion of proteins from liver homogenates
Equal amounts of protein (20 µg) were solubilized in a denaturation buffer (ammonium bicarbonate: 50 mM, pH 8; sodium deoxycholate: 1%; 30 µL final volume), heated to 95°C for 5 min. Disulfide bonds were reduced with 1 µg DTT (30 min at 37°C) and alkylated with 5 µg iodoacetamide (30 min at 37°C in the dark). Finally, trypsin was added at the indicated ratio, and the mixture was incubated at 37°C for 24 h. The digestion was stopped by acidifying the sample to pH<2.5 with 30 µL of a solution containing 3% ACN, 0.1% TFA and 0.5% acetic acid. Mixtures were left 10min. at room temperature and centrifuged 16,000g for 5min. The supernatant was desalted on C18-Empore Filter (Sigma). Digested peptides were eluted in a 80% ACN/0.1% TFA solution, dried in a SpeedVac, and resuspended in 20 μL of a 0.1% formic acid solution. 1 µL of this solution and 1 µL of standard peptide solution (5 fmol/µL) were spiked into 23 µL of 0.1% formic acid.
Solubilization and in-solution digestion of proteins from microsomal preparations
Equal amounts of protein from microsome preparations (20 µg) were loaded on desalting column Amicon 3 kDa (Millipore, Billerica, MA, USA). Samples were washed 3 times with 500 µL ammonium bicarbonate (50 mM, pH 8) and resuspended in 30 µL of the denaturation buffer (see above). The trypsin digestion was performed as above except for the reaction time which was 18 h.
MRM-MS analysis
One hundred nanograms of digested proteins (5 µL) were analyzed on a 5500 QTRAPTM hybrid triple quadrupole/linear ion trap mass spectrometer equipped with
a nanoLC AS2 cHiPLC nanoflex controlled by Analyst 1.6TM (Sciex, Concord, ON, Canada) and with a nanospray ionization source. Mass spectrometry analyses were conducted in positive ion mode with an ionspray voltage of 2300 V. Peptides were desalted on a 200 µm x0.5 mm chip trap column packed with ChromXP C18, 3 µm (Eksigent) at 2 µL/min of solvent A (formic acid, 0.1%). Then the peptides were eluted at a flow rate of 300 nL/min on a 75µm x 15cm chip column packed with ChromXP C18, 3 µm (Eksigent) on a 16 min linear gradient from 5 to 25% of solvent B (ACN, 3%; FA, 0.1%), yielding a total cycle run time of 2.9 sec. The nebulizer gas was set at 8 (Gas 1), curtain gas at 20, heater at 150°C and the declustering potential (DP) at 70 V.
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The selection of the most suitable peptides for sensitive and selective protein detection was performed after in silico digestion of the CYP3A4 and Calnexin proteins using the open source Skyline v2.5 program (MacCoss Lab, Seattle, WA, USA). Peptides containing methionine and cysteine were eliminated as well as non-uniques peptides. Theoretical MRM transitions were generated by including y-ions from +2 charge state precursor with mass above 300 Da and below 1500Da. The predicted peptides were then validated through trypsin digestion of microsomal proteins. MRM-MS analyses using a full scan MS-MS mode for each detected MRM transition were performed for sequence verification of the hypothetical peptide after database search with Mascot search engine (Matrix Science, London UK). Two peptides per protein were selected based on peak shape and intensity. MRM-MS analyses were performed using the 3 most intense transitions for each of the target peptides. The MRM transition with the highest area counts was subsequently used for protein quantification, while the 2 remaining transitions served as qualifier transitions to confirm peptide retention times and the fragment ion ratios. A blank solvent injection was run between biological samples to prevent sample carryover and the samples were injected in random order. Samples were analyzed in duplicate. Samples containing 5fmol of digested BSA were injected periodically in order to confirm system stability.
Stable-isotope-labeled standard (SIS) peptides
Purified synthetic peptides containing [13C
6,15N2]Lys and [13C6,15N4]Arg were
reconstituted in 0.1% formic acid to a final concentration of 500,000fmol/µL. A solution containing 0.2fmol/µL of each peptide was prepared from the stock solutions and used to reconstitute the samples after tryptic digestion for absolute quantification. The purity of AQUA peptides was >95% as evaluated by HPLC.
Precision and accuracy
Intra-day assay validation was assessed by the replicate analyses of 5 homogenate and 5 microsomal samples prepared independently (1 sample/day) from a single liver donor. Inter-day assays were then performed with a selected homogenates stored at -20°C for 1, 7 and 14 days. In these assays, CYP3A4 and Calnexin proteins were quantified through MRM-MS measurement as described above. Intra- and inter-variation of results are expressed as % coefficient of variation (%CV) in Supporting Information Tables 2 and 3.
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Enzymatic activity assay
The CYP3A4 enzymatic assay was performed as previously described [521]. Briefly, liver homogenates and microsomes (25 µg) were incubated in the presence of 25 µM chenodeoxycholic acid (CDCA) for 1 h. The formation of the CYP3A4 product hyocholic acid (HCA), was then quantified through LC-MS/MS using an API4000 instrument equipped with an electrospray ionization source (Applied Biosystems, Concord, Canada) as extensively described elsewhere [522].
Statistics
All data are presented as mean ± standard deviation (S.D.). Shapiro-Wilk test was performed to evaluate if the distribution of variables are close to a normal distribution. Correlation between homogenates and microsomes groups among all subjects was analyzed by Spearman's rank correlation coefficient and comparisons between two groups were performed using Wilcoxon test. All analyses were done with SPSS 18.0 software (SPSS Inc, Chicago, IL, USA).
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RESULTS AND DISCUSSION
Protein and peptide selection
To develop a sensitive assay for quantification of CYP3A4 and the reference protein calnexin by MRM-MS, four peptides, two for each protein, were selected using Skyline v2.5 (MacCoss Lab) based on peak shape and intensity and assigned so that they are not adjacent to any other human proteins. Thus, the EVTNFLR and LSLGGLLQPEKPVVLK were selected for CYP3A4 quantification and EIEDPEDR, AEEDEILNR for the reference protein calnexin (Fig. 1). Corresponding proteotypic peptides containing isotopically coded amino acids [13C
6,15N2]Lys or [13C6,15N4]Arg were then synthesized to serve as internal
standard for CYP3A4 and calnexin quantification. Representative chromatograms of transition ions of the stable-isotope labeled peptides in human microsomes and homogenates for each targeted peptide are illustrated in Fig. 1 and Supporting Information Fig. 1. A sample fragment ion spectra is also illustrated in supplemental Fig. 2, while all skyline files are available on the Panorama website (https://panoramaweb.org/labkey/cyp3A4.url).
Optimization of trypsin digestion
Since the MRM analysis quantifies proteotypic peptides, and not intact proteins, quantification values are crucially dependent on the efficiency of the trypsin digestion. Optimal trypsin digestion procedures for the determination of microsomal proteins from human liver have been previously reported [59], but in complex biological samples such as tissue homogenates, digestion peptides are generated at lower rates most likely due to higher steric hindrance [523]. Since the MRM analysis quantifies proteotypic peptides, and not intact proteins, quantification values are crucially dependent on the efficiency of the trypsin digestion. We therefore optimized the two most important parameters for this enzymatic reaction, namely the enzyme (i.e. trypsin) to substrate (i.e protein) ratio and digestion time, using liver homogenates. Four digestion ratios (protein:trypsin, 3.1:1, 6.3:1, 12.5:1, 25.0:1) were tested with a digestion duration varying from 12 to 24 h. As shown in Fig. 2, the most efficient digestion time was 24 h for all peptides, however longer incubation is also to be considered to confirm a complete peptide release. Under this condition, the higher concentrations for both CYP3A4 peptides were observed with the 3.1 to 1 protein:trypsin ratio, and the digestion efficiency was reduced with increasing ratios (Fig. 2A and B). The calnexin target peptides were less sensitive to this parameter (Fig. 2C
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and D), thus we selected the 24 h incubation with the 3.1:1 protein/trypsin ratio as the optimum digestion condition for the subsequent analyses of liver homogenates. However, we cannot exclude that post-translational modifications or incomplete digest of the protein could affect the selected peptides for subsequent quantification.
Experimental validation of the targeted peptides
To evaluate the sensitivity and dynamic range of CYP3A4 quantification by multiplexed MRM detection, each selected peptide was analyzed in microsomal liver samples with 1 fmol per 10µg of digested proteins of the corresponding internal standard using three sets of MRM transitions (Fig. 1 and Supporting Information Fig. 2). The MRM transition that gave the highest area counts was subsequently used for the quantitation, with the other two transitions acting as qualifier transitions to confirm peptide retention times and the fragment ion ratios. The calibration curves of four peptides in the trypsin digested liver samples (100 ng microsomes) showed a strong linearity (r2>0.98). The
concentration range used in these duplicate analyses was selected based on previous reports where endogenous CYP3A4 levels were determined [524, 525]. For the two calnexin peptides and the CYP3A4 EVTNFLR peptide, the linearity was obtained with the complete concentrations range (Fig. 3A, C and D), while the CYP3A4 LSLGGLLQPEKPVVLK peptide, which is the most hydrophobic one, exhibited a limited linear concentration range (Fig. 3B). This peptide was therefore excluded for subsequent analyses. The EVTNFLR peptide was selected to quantify CYP3A4 concentration in other