Absolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach

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1 CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 42, Issue 1, January 2014 Online English edition of the Chinese language journal Cite this article as: Chin J Anal Chem, 2014, 42(1), RESEARCH PAPER Absolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach LIU Xi-Dong 1, ZHU Jun 2, CONG Yu-Ting 1, HU Liang-Hai 1, *, YE Ming-Liang 2, GU Jing-Kai 1, ZOU Han-Fa 2 1 School of Life Sciences, Jilin University, Changchun , China 2 Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian , China Abstract: A strategy for the absolute quantitation of metabolic enzymes in rat liver microsomes was developed based on shotgun-based proteomics approach. Rat liver microsomes were digested with trypsin, and drug metabolizing enzymes were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using standard curves based on custom synthesized specific peptides for each enzyme. The assays for cytochrome P450 (CYP450) and uridine diphosphoglucuronosyl transferase (UGT) showed good linearity (r > 0.995) with lower limits of quantitation of 10 nm. A synthetic isotope labeled specific peptide was then used as internal standard to determine UGT1A1 by stable isotope dilution method. The labeled peptide behaved similarly to the unlabeled peptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1 concentration was detected by the labeled peptide method to be 18.2 pmol mg 1 protein compared with 17.3 pmol mg 1 protein obtained by the standard curve method. Although the consistent results were achieved by the two methods, the stable isotope dilution method was more convenient and more suitable for high throughput determinations in complex systems. Key Words: Liquid chromatograhpy-mass spectrometry; Cytochrome P450; Uridine-diphosphate glucuronosyl transferase; Multiple reaction monitoring; Liver microsome 1 Introduction Drugs can be metabolized and eliminated from the body via the production of active and inactive metabolites, and the formation of metabolized drugs has a significant influence on the therapeutic effect and safety of a drug [1,2]. Among the drug metabolizing enzymes (DMEs), cytochrome P450 (CYP or P450) and uridine diphosphoglucuronosyl transferase (UGT) are the most important and responsible for the metabolism of most drugs. Therefore, the quantitative analysis of DMEs and UGT is an important aspect of drug metabolism research, which provides a useful contribution to drug development, drug-drug interaction studies and preclinical research. Previously, DMEs were commonly determined using reverse transcription polymerase chain reaction (RT-PCR) [3,4], 2-D electrophoresis [5], western blotting [6,7] and chemical probes [8,9]. However, these methods have a number of disadvantages including the fact that the amount of mrna does not necessarily reflect the protein level and high amino acid homology results in cross reactivity and associated poor specificity. With the development and application of massspectrometric techniques in proteomics [10,11], the quantitative analysis of peptides and proteins in complex systems has been realized, which provides a theoretical and experimental basis for the quantitation of DMEs. In this study, specific peptides produced by trypsinization of DME proteins were identified, Received 29 July 2013; accepted 22 October 2013 * Corresponding author. lianghaihu@jlu.edu.cn This work was supported by the National Natural Science Foundation of China (No ), the Program for New Century Excellent Talents in University of China (No.NCET ), and the Open Project from State Key Laboratory of Proteomics, China. Copyright 2014, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: /S (13)

2 custom synthesized and used to identify the presence of the proteins in a sample of rat liver microsomes by comparison of their fragmentation patterns in liquid chromatography tandem mass spectrometry (LC-MS/MS). They were then used as external standards to carry out the simultaneous quantitative analysis of CYP450 and UGT in rat liver microsomes. Subsequently, a specific peptide of UGT1A1 was isotope labeled and used as an internal standard (IS) to determine UGT1A1 in the same sample by the isotope dilution method. Compared with previous methods, direct LC-MS/MS is less time consuming, more accurate, more sensitive, and allows high throughput analysis. It will provide an important tool for future research on drug metabolism enzymes. 2 Experimental 2.1 Instruments and reagents LC-MS/MS was carried out on an Agilent 1100 Series HPLC system (Agilent Technologies, USA) with an SIL-20AC autosampler (Shimadzu, Japan). The HPLC system was coupled to an API 4000 mass spectrometer (AB SCIEX, USA) equipped with electrospray ionization (ESI) source operated in the positive ion mode and Analyst Software. Chromatography was performed on a peptide C18 column (50 mm 2.1 mm, 2.1 m, AB SCIEX, USA) maintained at 40 C. The CYP450 and UGT specific peptides GNPESSFNDANLR, YIDFVPIPLPR, VHEEIEQVIGR, FADIVPTNIPHMTSR, IILNELAQR, LLDVWTYELPR and SVFDQDPFLLR and the UGT1A1 isotope labeled peptide SVFDQDPFLLR* (C 13, N 15 ) were custom synthesized by Shanghai Chutai Biological Technology Co., Ltd. Acetonitrile was chromatographically pure (Sigma-Aldrich, USA), formic acid was analytically pure (Beijing Chemical Works) and water was produced using a Milli-Q ultrapure water system. 2.2 Methods LC-MS/MS conditions Gradient elution was carried out by using acetonitrile: 0.1% formic acid (2:98, V/V) as solvent A and acetonitrile:0.1% formic acid (98:2, V/V) as solvent B with the following program: 0 1 min, 95% A; 1 9 min, 95% 60% A; 9 11 min, 60% A; min, 60% 95% A; min, 95% A. The injection volume was 5 L and the flow rate was 0.5 ml min 1. The electrospray (ESI) voltage and heater gas temperature of the ESI source were set at 5000 V and 450 C respectively. Nitrogen was used as nebulizer gas (45 Psi), auxiliary gas (40 Psi) and curtain gas (20 Psi). MRM transitions, collision energies (CEs) and declustering potentials (DPs) are given in Table Preparation of specific peptide calibration standards Stock solutions of specific peptides (5 g L 1 ) were prepared separately in water and diluted with 0.1% formic acid solution to give standard solutions with concentrations of 100 pmol L 1 and 100 fmol L 1. Finally, a series of mixed standard solutions of the seven peptides were prepared in 5 ml volumetric flasks Preparation of rat liver microsomes A rat was sacrificed by decapitation and exsanguinated before its liver was removed, weighed and cooled in an ice-water bath. The liver was washed repeatedly with normal saline at 4 C and cut into 2 mm 3 pieces. After added a volume (ml) of 50 mm Tris-HCl buffer solution (150 mm NaCl, 5 mm EDTA, 1% NP 40 and protease inhibitor cocktail, ph 7.4) equal to 3 times the liver weight, the liver was homogenized by a homogenizer. Then the homogenate was centrifuged at 9000 g for 20 min at 4 C and the supernatant filtered through 4 tier gauzes. The filtrate was retained as the S9 component. The S9 fraction was centrifuged at g for 3 h at 4 C to obtain a pellet of liver microsomes which was suspended in Tris-HCl buffer and stored at 80 C before analysis Preparation of isotope labeled specific peptide calibration standards Aliquots (75 L) of rat liver microsomes were added to 2 tubes. Then three incubations were carried out sequentially as Table 1 Amino acid sequences and MRM analytical parameters of specific peptides for determination of drug metabolizing enzyme isoforms Isoforms Sequence Position Molecular weight Q1 Mass (amu) Q3 Mass (amu) DP (Volt) CE (ev) CYP2D3 GNPESSFNDANLR CYP2C70 YIDFVPIPLPR CYP2A1 VHEEIEQVIGR CYP2D26 FADIVPTNIPHMTSR UGT2B1 IILNELAQR UGT2B2 LLDVWTYELPR UGT1A1 SVFDQDPFLLR UGT1A1 SVFDQDPFLLR*

3 follows: (1) Incubated at 37 C for 1 h after added 425 L buffer solution I (50 mm NH 4 HCO 3, 8 M urea) and 20 L 300 mm dithiothreitol solution; (2) Incubated at 25 C for 40 min after added 60 L 300 mm freshly prepared iodoacetamide solution; (3) Incubated at 37 C for 6 h after added 2000 L buffer solution II (50 mm NH 4 HCO 3 ) and 12.5 L 0.5 g L 1 trypsin solution. The reaction was terminated by adding 100 L of 10% TFA solution. The impurity in the sample was removed by solid phase extraction (SPE) and the peptide in the sample was eluted by 1 ml acetonitrile-water-tfa solution (80:20:0.1, V/V). Finally, the treated sample above was centrifuged, freeze-dried, and diluted by a 500 L of 0.1% formic acid solution to obtain the peptide matrix labeled M. A stock solution of isotope labeled synthetic peptide (SVFDQDPFLLR*) (5 g L 1 ) in water was diluted with a 0.1% formic acid solution to give a concentration of 100 mm. Then the diluted solution above was further diluted with solution M to give a series of calibration standards of isotope labeled specific peptide Enzymolysis and determination of DMDs in rat liver microsomes Preparation of liver microsomes sample followed the steps described in Section Firstly, an aliquot of rat liver microsomes (15 L) was mixed with 85 L of buffer solution I (50 mm NH 4 HCO M Urea) and 4 L of 300 mm dithiothreitol solution in a 1.5 ml Eppendorff tube. Then the mixture was incubated at 37 C for 1 h. Secondly, 12 L freshly prepared 300 mm iodoacetamide solution was added and then the tube was incubated at 25 C for 40 min. Thirdly, 400 L buffer solution II (50 mm NH 4 HCO 3 ) and 2.5 L 0.5 g L 1 trypsin solution were added and then the tube was incubated at 37 C for 6 h. The reaction was terminated by adding 20 L of 10% TFA solution. The product was subjected to SPE as described above. Finally, the sample was diluted with 90 L of 0.1% formic acid solution and 10 L of 100 μm isotope labeled peptide solution and analyzed by LC-MS/MS. 3 Results and discussion 3.1 Selection of specific peptides and LC-MS/MS conditions First, specific peptides representative of DMD enzymes were selected through analysis of the amino acid sequence of CYP450 and UGT. In parallel, a sample of liver microsomes was examined by high resolution mass spectrometry-ltq- Orbitrap [12] and the presence of the specific peptides were confirmed by real sample analysis and application of peptide selection principles [13]. For example, as shown in Figs.1A and 1B [12], the specific peptides of UGT1A1 and SVFDQDPFLLR* were identified by the MS and MS/MS spectra. Due to the difference in fragmentation patterns generated by different types of instruments [14], the synthetic peptides were used to optimize MRM parameters. For example, the double charged precursor ion at m/z of SVFDQDPFLLR was selected in the Q1 MS scan mode (Fig.1C) after which the high intensity fragment ion at m/z was selected by adjustment of the collision energy (Fig.1D). Finally, this MRM transition was selected for the quantitative analysis of target peptides. Since the specific peptides maintain a double or triple charge during the ionization process, formic acid was also added to the mobile phase to provide the protons. A short 50 mm column was used to shorten the analysis time and a gradient elution was employed to avoid interference from target peptides. Chromatograms of the synthetic peptides selected are shown in Fig.2. Fig.1 MS spectra (A) and MS/MS spectra (B) of UGT1A1 specific peptide in a rat liver microsome sample as determined using LTQ-Orbitrap; MS spectra (C) and MS/MS spectra (D) of the same peptide determined using LC-MS/MS

4 pmol mg 1 protein. As shown in Fig.5, the results of the two methods of analysis are in close agreement. However, isotope-dilution analysis is more suitable for high throughput quantitation of DMEs and has the potential for wider application in DME research. Fig.2 HPLC-MRM chromatograms of specific peptides of CYP450s and UGTs in rat liver microsomes 3.2 Preparation of standard curves for specific peptides Linearity of each peptide was established by weighted (1/x 2 ) least squares linear regression [15] of their respective calibration curves. As shown in Table 2, the correlation coefficients were all > and the lower limit of quantitation (LLOQ) was 2 nm. Fig.3 HPLC-MRM chromatogram of 300 nm mixed standard solution of unlabeled and labeled UGT1A1 specific peptide 3.3 Investigation of isotope labeled peptide as internal standard Standard curves can be used for the absolute quantitation of specific peptides but are not suitable for the simultaneous quantitation of peptides in a complex matrix. However, this can be done using isotope dilution analysis. In this study, C 13 and N 15 labeled arginine were used to synthesize a target peptide specific to UGT1A1, and by the method, due to the same physicochemical properties as the unlabeled peptide, the potential matrix effects could be eliminated. As shown in Fig.3, the stable isotope labeled peptide and unlabeled peptide in 0.1% formic acid solution exhibited a consistent chromatographic retention and peak areas, indicating the stable isotope labeled peptide was suitable for use as an IS. Fig.4 Standard curve for stable isotope labeled UGT1A1 specific peptide 3.4 Determination of DMEs in rat liver microsomes By using calibration curves of specific target peptides, the concentrations in liver microsomes of CYP2D3, CYP2C70, CYP2A1, CYP2D26, UGT2B1, UGT2B2 and UGT1A1 were found to be 11.77, 15.80, 26.82, 18.30, 11.47, and pmol mg 1 protein, respectively (Fig.5). The concentration of UGT1A1 determined by isotope-dilution analysis was Fig.5 Concentrations of seven drug metabolizing enzymes a rat liver sample Table 2 Linear ranges and standard curve equations for specific peptides Peptide sequence Linear range (nm) Standard curve equation Correlation coefficient GNPESSFNDANLR y = 820x YIDFVPIPLPR y = 904x VHEEIEQVIGR y = 1190x FADIVPTNIPHMTSR y = 1170x IILNELAQR y = 509x LLDVWTYELPR y = 222x SVFDQDPFLLR y = 835x

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