Distinction between Human Cytochrome P450 (CYP) Isoforms and

Oct 2, 2008 - To whom correspondence should be addressed. Tel.: +49 234 32/ 29265 or 28444. Fax: +49 234 32 14554. E-mails: (G.R.) gorden.redlich@rub...
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Distinction between Human Cytochrome P450 (CYP) Isoforms and Identification of New Phosphorylation Sites by Mass Spectrometry Gorden Redlich,*,† Ulrich M. Zanger,‡ Stephan Riedmaier,‡ Nicolai Bache,§ Anders B. M. Giessing,§ Martin Eisenacher,| Christian Stephan,| Helmut E. Meyer,| Ole N. Jensen,§ and Katrin Marcus*,† Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, Universitaetsstr. 150, ZKF, D-44801 Bochum, Germany, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, D-70376 Stuttgart, Germany, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark, and Medical Proteomics & Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum, Universitaetsstr. 150, ZKF, D-44801 Bochum, Germany Received March 27, 2008

In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes. Keywords: cytochrome • P450 • phosphorylation • CYP • quantification • differentiation • distinction • discrimination • distinguish • mass spectrometry

Introduction The Cytochrome P450 (CYP) super family consists of hemedependent enzymes that catalyze a vast number of oxidative biotransformations of exogenous and endogenous substances. They are found in all five biological kingdoms and over 7700 different CYP genes have been described to date1 [http:// drnelson.utmem.edu/CytochromeP450.html]. Whereas bacterial P450s are usually cytosolic, all eukaryotic P450 enzymes

* To whom correspondence should be addressed. Tel.: +49 234 32/ 29265 or 28444. Fax: +49 234 32 14554. E-mails: (G.R.) gorden.redlich@ rub.de, (K.M.) [email protected], www.medizinisches-proteom-center.de. † Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum. ‡ Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology. § Department of Biochemistry and Molecular Biology, University of Southern Denmark. | Medical Proteomics & Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universitaet Bochum.

4678 Journal of Proteome Research 2008, 7, 4678–4688 Published on Web 10/02/2008

Table 1. Main Human Drug-Metabolizing CYPs3 CYP1A1,a CYP2B6, CYP2D6, a

CYP1A2, CYP2C8, CYP2E1,

CYP1B1,a CYP2C9, CYP3A4,

CYP2A6, CYP2C19, CYP3A5

Not detectable in human liver microsomes.

are membrane-anchored by their hydrophobic N-terminus either in the endoplasmic reticulum (ER) or in the mitochondria.2 Twelve of the 57 human CYP isoforms are known to play a major role in the metabolism of many xenobiotics including most clinically used drugs (Table 1).3,4 Variation in the expression and/or function of drug metabolizing P450s affects drug pharmacokinetics and has clinical relevance in particular for drugs with narrow therapeutic windows. Reasons for the observed large inter- and intraindividual variability of CYP expression and activity can be attributed to environmental influences, physiological and genetic factors including drug-drug interactions, sex-dependent expression, and genetic polymorphisms.4,5 10.1021/pr800231w CCC: $40.75

 2008 American Chemical Society

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Identification of CYP Isoforms and Phosphorylation Sites by MS Quantitative relationships between CYP protein expression in biological tissues and their functional phenotypes, genotype, and physiological or pathophysiological factors are required in the context of preclinical drug development and in vivo extrapolation6 as well as for mathematical modeling approaches in systems biology.7 The quantification methods that are most widely used today are immunological techniques such as ELISA and Western blotting8 which rely on the availability of specific antibodies. Although tremendous efforts have been made to develop isoform-specific monoclonal9 or anti-peptide antibodies,10 the simultaneous and comparative quantification of CYP enzymes is still a very difficult task because of their high similarities both in sequence and in molecular weight. For example, recent data on the relative expression levels of CYP3A4 and CYP3A5 have remained controversial.11-13 Similar problems persist for other larger subfamilies including CYP2C and CYP4F. Mass spectrometry (MS) has revolutionized protein science over the last years14 and has the potential to provide fast, sensitive, and specific analysis of P450 enzymes, although it is not as sensitive as immune detection with specific antibodies. Further, mass spectrometry offers high multiplexing capability and the possibility of relative or absolute quantification of proteins.15 MS analysis is typically performed at the peptide level, that is, after enzymatic proteolysis of proteins, and therefore, only distinct peptides can be used for unambiguous identification of the corresponding isoforms. In case of P450 enzymes, this is a demanding task due to the high sequence similarity between isoforms. For example, the sequence similarity of CYP2C9 and CYP2C19 is 91%, which results in the generation of rather few isoform-specific peptides upon digestion of the proteins by trypsin. Importantly, MS-based technologies have even further advantages in that they are principally able to not only detect simultaneously different isoforms but also different posttranslationally modified forms of a protein.16 Among many known types of post-translational modifications, phosphorylation is arguably one of the most important. Phosphorylation was previously suggested to affect activity17 and mitochondrial import rate18 as well as degradation19 of some Cytochrome P450 forms. Only a few phosphorylation sites were identified among human CYPs using in vitro kinase assays, namely, in CYP2E1,20 CYP3A4,21 and possibly in CYP1A2.22 Thus, CYP phosphorylation is still poorly analyzed and far from being understood, in particular with respect to its in vivo relevance for enzyme turnover rates and function. Traditional approaches for phosphoprotein analysis use antibodies that specifically recognize a phosphorylated epitope of a protein. A major drawback of this method is the timeconsuming process that often takes up to 1 year to generate and validate these antibodies.23 A less site-specific strategy uses antibodies that recognize a kinase consensus motif. But since more than 500 different human kinases are known so far, only a subphosphoproteome can be analyzed by this.24,25 To solve this disadvantage, antibodies that recognize a phosphoresidue independently of the surrounding sequence can be applied. However, only anti-phosphotyrosine in contrast to phosphoserine/-threonine antibodies are available that show the necessary specificity.23 This limits the application of this approach since the phosphorylation ratio of the amino acids serine, threonine and tyrosine is approximately 1800:200:1.26 Today mass spectrometry is the most powerful method for site-specific identification of protein phosphorylation on a

proteomic scale. It has to be mentioned that protein phosphorylation is typically substoichiometric. In contrast to antibodybased phosphoprotein detection, enrichment methods prior to mass spectrometric analysis have to be applied to cover the corresponding phosphopeptides.27,28 The use of TiO2 material turned out to be very efficient for such a purpose.29,30 In this study, we present the successful application of a mass spectrometry-based proteomic strategy to differentiate between very homologous CYP isoforms in a complex protein mixture that could not be simultaneously distinguished by other methods so far. Further, it is shown that MS/MS sequencing can be used to evaluate the amounts of CYP isoforms in a semiquantitative manner. The second aim of this study was the identification of new CYP phosphorylation sites in vivo. By the use of the TiO2 technique for phosphopeptide enrichment followed by mass spectrometric sequencing, eight CYP phosphorylation sites could be localized, seven of which have never been described before. The data can serve as a basis for targeted proteomic approaches to relatively or absolutely quantify the CYP isoforms covered in this approach as well as the new identified phosphorylation sites.

Materials and Methods Human Liver Tissue. Human liver tissue was obtained as nontumorous tissue surrounding surgically removed liver tumors or material surgically removed for other reasons from four different individuals of Caucasian origin. The tissue was immediately frozen and stored at -80 °C until subcellular fractions were prepared by standard differential ultracentrifugation as already described.31 Briefly, approximately 1 g of tissue was homogenized in 1 mM DTT, 10 mM HEPES (pH 7.4) and 0.15 mM KCl. Protease and phosphatase inhibitors (Complete with EDTA, Roche Applied Science; Phosphatase-inhibitor cocktail set II, EMD Biosciences) were added to all applied buffers following supplier’s instructions. The homogenate was centrifuged at 15 000g for 30 min to remove the nuclei and cell debris. The supernatant was centrifuged at 105 000g for 60 min to obtain the microsomal pellet that contained most of the CYPs. The supernatant with the cytosolic proteins was discarded. The microsomal pellet was washed once with 0.1 M sodium pyrophosphate buffer (pH 7.5). After recentrifugation at 105 000g for 60 min, the pellet was resuspended in 0.1 M sodium pyrophosphate buffer (pH 7.4) and immediately frozen at -80 °C in aliquots after determination of the protein concentration by Bradford-Assay.32 The study was approved by the ethics committee of the Medical Faculty of the Charite´, Humboldt-University, Berlin, Germany. Gel Electrophoresis. One-dimensional gel electrophoresis was performed according to Laemmli.33 In brief, microsomal fractions were dissolved in SDS sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 0.05% bromophenol blue, 100 mM DTT), heated to 95 °C for 5 min, and applied to a 4-20% polyacrylamide precasted separation gel (PAGEr, Lonza Copenhagen ApS). Electrophoresis was carried out at 50 V for 10 min and further 200 V until the bromophenol blue front reached the bottom of the gel. Separated proteins were visualized by Coomassie staining. Fixation and staining was done in one step for 45 min with 0.25% Coomassie Blue R250 in methanol/acetic acid/water (45:10:45), followed by rinsing for 15 min in methanol/acetic acid/water (45:10:45) and destaining in water overnight. Protein Digestion. In-gel digestion was performed according to Shevchenko et al.34 Cut protein bands were washed, followed Journal of Proteome Research • Vol. 7, No. 11, 2008 4679

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Figure 1. Overview of the experimental workflow. The solubilized proteins of human liver microsomes were separated by 1D-SDSPAGE. After Coomassie staining, the accordant CYP bands were excised and digested with trypsin. Phosphopeptides were enriched by the use of TiO2 microcolumns followed by desalting the flow through and elution fractions with reversed-phase Poros material: R2 for the nonphosphopeptide fraction, R3 for the phosphopeptide fraction. Peptide and phosphorylation site analysis was done by nanoLC-ESI-MS/MS on a 4000QTrap (ABI/ MDSSciex) system. Proteins were identified by MASCOT and the use of the current Swiss-Prot/Uniprot database version.

by a reduction step using 10 mM DTT in 100 mM NH4HCO3, pH 7.8 (56 °C, 45 min). Alkylation of cysteines was achieved with a solution of 55 mM iodoacetamide in 100 mM NH4HCO3 (room temperature, in the dark, 30 min). After washing the gel pieces, they were reswelled 45 min on ice with 12.5 ng/µL trypsin (modified, sequence grade, Promega) in 50 mM NH4HCO3. The supernatant was replaced by 50 mM NH4HCO3 without trypsin and the gel particles were incubated at 37 °C overnight. Peptides were extracted two times with 50% acetonitrile (ACN), 50 mM NH4HCO3, pH 7.8 Phosphopeptide Enrichment and Desalting. TiO2 microcolumns29 were used to enrich the phosphopeptides according to the published protocols.35,36 The workflow is shown in Figure 1. In brief, GELoader Tips (Eppendorf) were sealed with a C8 plug (C8 Empore disk, 3M) and packed with TiO2 material (Titansphere, 5 µm, GL Sciences). Dried peptides were solubilized in loading buffer (1 M glycolic acid, 5% TFA, 80% ACN). After loading, the column was successively washed with loading buffer, washing solution (80% ACN, 1% TFA) and ultra pure water while collecting each flow through. Phosphopeptides were eluted using aqueous ammonia (1.5% ammonium hydroxide, pH 10.5) followed by 30% ACN to elute the bound peptides from the C8 disk. Subsequently, peptides were acidified with FA. Flow through and elution fractions were desalted using reversed-phase microcolumns as already described.37,38 GELoader Tips were sealed with a C8 plug and packed with R2 (for the TiO2 flow through fraction) or R3 material (for TiO2 elution) (Reversed-phase Poros media, Perseptive Biosystems). Dried peptides were resolubilized using 5% FA, loaded onto the column and washed with 5% FA. Elution was done using 5% FA + 80% ACN followed by drying of the peptides. LC-ESI-MS/MS. Samples were resolubilized in 5% formic acid. Tryptic peptides were separated on a nanoliter-flow Ultimate 3000 HPLC system (Dionex LC Packings, Idstein, Germany). After injection, peptides were trapped and desalted on a homemade 1-cm fused-silica precolumn at a flow rate of 4680

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Redlich et al. 3 µL/min in 5% FA (100 µm i.d., ReproSil-Pur C18 AQ, 3-µm, Dr. Maisch GmbH). The concentrated sample was then eluted onto a homemade 8-cm fused-silica separation column (50 µm i.d., ReproSil-Pur C18 AQ, 3-µm, Dr. Maisch GmbH) with a flow rate of 200 nL/min. Mobile phases were A, 5% formic acid and B, 5% FA, 80% ACN. A mobile phase gradient was used to elute the peptides: 12 min 0% buffer B, 58 min gradient from 0% to 50% buffer B, 2 min gradient from 50% to 100% buffer B, 5 min 100% buffer B followed by 13 min 0% buffer B. The nanoliter-flow HPLC system was interfaced to a 4000QTrap mass spectrometer (MDS Sciex, Applied Biosystems)39 equipped with a NanoSpray II source and a PicoTip emitter needle (FS360-20-10-N-20-C12, New Objective). The MS scanning cycle was set up as follows: an enhanced MS scan with 4000 amu/s (EMS) was acquired from 400 to 1600 m/z, two spectra were summed, leading to a scan time of 0.78 s. This was followed by an enhanced resolution scan (ER) with 250 amu/s to determine the charge state of the five most abundant peptide ions, which were selected for enhanced product ion scanning (EPI) or MS/MS. Two scans were summed leading to a scan time of 1.58 s. After two occurrences, a precursor was excluded from MS/MS for 45 s. EPI scans were acquired with a scan rate of 4000 amu/s for 45 ms from 100 to 280 m/z and for 306 ms from 275 to 1500 m/z with a 0.55 s scan time for one EPI. The total cycle time was 4.73 s and the declustering potential was set to 60. Gs1 values between 16 and 20 and needle voltages between 2600 and 3000 V were used. Peptide/Protein Identification. The MS data files (recorded with Analyst Software Version 1.4.2, ABI/MDSSciex) obtained by LC-MS/MS analysis of the TiO2 flow through (nonphosphorylated peptides) and LC-MS/MS analysis of the TiO2 eluate (phosphopeptides) were converted to the mascot generic format (mgf). Default precursor charge states from 1+ to 4+ were selected. The precursor mass tolerance for grouping was set to 1, with a maximum of 10 cycles between groups and a minimum number of 1 cycle per group. MS/MS spectra were rejected if less than 10 peaks were present. No peaks were removed by the algorithm. All MS/MS data were centroided and deisotoped. Combined mgf-files were sent to an in-house MASCOT server (Version 2.2, Matrix Science40) for automated peptide identification using the Swiss-Prot/Uniprot database (Version 54.4) with human taxonomy restriction. The following Mascot settings were used: carbamidomethyl was specified as a fixed modification; methionine oxidation, serine, threonine and tyrosine phosphorylation were specified as variable modifications. One missed cleavage was allowed. The precursor mass tolerance was set to 1.2 Da and the fragment mass tolerance to 0.6 Da. A protein was rated as a correct match without manual revalidation, if at least two unique peptides with a score higher than 30 and an expect value lower than 0.05 were assigned to it. Spectra with MASCOT scores from 23 to 30 and/or expect values from 0.05 to 0.99 as well as all phosphopeptide assignments and all protein identifications with only one unique peptide were manually validated by inspection of the accordant MS/MS spectra to eliminate false positive matches. Spectra assigned to a peptide sequence with Mascot internal expect values higher than 0.99 were rejected without any inspection. The phosphorylation prediction tool NetPhos41 (Version 2.0) was applied to judge the mass spectrometry assignment of phosphorylation sites.42 A prediction probability value between 0.9 and 1.0 for a candidate phosphorylation site is an indication, but not a proof, that the phosphorylation site assignment is valid.

Identification of CYP Isoforms and Phosphorylation Sites by MS

Figure 2. Electrophoretic separation of microsomal protein. The microsomal fractions (20 µg of protein per lane from four different human donors) were separated on a 4-20% Tris-glycine gel followed by Coomassie staining. The red box indicates the isolated region containing most of the CYPs.

Results and Discussion Distinction between CYP Isoforms. To reduce sample complexity, microsomal proteins were separated by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The Coomassie stained gel of the separated microsomal protein fractions of four different liver donors is shown in Figure 2. The CYPs can be detected over a narrow mass range from 46 to 58 kDa, although they should have nearly the same mass of around 56 kDa. After tryptic in-gel digestion, a total of 22 different CYPs could be distinguished by MS (Table 2). Among them, all major drug-metabolizing CYP isoforms3,43 (Table 1), namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, were identified. Notably, we found no indication of the presence of several CYPs regarded as extrahepatic including CYP1A1, CYP1B1 and CYP2F1. Table 2 describes these findings with detailed information about the identified CYP isoforms for each of the four donor samples. Additionally, the number of all peptides and the number of unique peptides assigned to each protein are displayed. Further, the theoretical number of unique peptides in each protein sequence is given. The latter is including tryptic peptides with no missed cleavages, a length of 5-20 amino acids and a mass between 800-4000 Da, since larger peptides are often not efficiently detected or sequenced by standard LCESI-MS/MS. When interpreting Table 2, it has to be taken into account that in some cases the number of identified unique peptides is higher than the number of theoretical unique peptides in the sequence. This is due to the additional identification of peptides with missed cleavage sites or peptides that were longer than 20 amino acids. Again, these were not included in the list of theoretically generated unique peptides. Further, it has to be mentioned that it is highly recommended to use the Swis-Prot/Uniprot protein database for automated identification of Cytochrome P450 isoforms from peptide MS/ MS spectra because it is redundancy-free and contains one validated entry per CYP isoform. The current IPI database for example, that also is redundancy-free, contains in some cases many variants of the same CYP isoform, and especially in case of CYP2D6, the wild-type form is even not included in the database. This leads to hardly interpretable results. Some CYPs can be distinguished from others by a large number of unique peptides as for example CYP1A2. Most of

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the theoretical 18 unique peptides can be identified by MS/ MS due to the high abundance of CYP1A2 in microsomes of all 4 donors (Table 2). Even more, namely, 27 theoretical unique peptides can be calculated for CYP2J2, but due to the low abundance of this isoform in the samples of all 4 donors, it was only identified in donors 1 and 3 with 2 and 1 unique peptides, respectively. CYP2A6 can unambiguously be identified by only four tryptic peptides with regard to the distinction from the highly homologous CYP2A7 (94% sequence similarity) and CYP2A13 (93% sequence similarity). Regardless, it could be identified in the samples of all 4 donors. CYP2A7 and CYP2A13 could not be definitely identified at all. A few CYPs were detected by several but just one unique peptide due to their high sequence similarity. Only CYP7B1 in donor 1 and CYP2J2 in donor 3 were identified by one peptide at all. Of course, those peptides must be unique for the appropriate isoform to allow for their explicit identification. Table 3 summarizes the peptides used for single peptide-based identifications. The accordant, manually validated spectra can be found in the supplemetary material I in Supporting Information. We also demonstrated, that the highly similar isoforms of the CYP3A, CYP2C and CYP4F subfamilies (namely, CYP3A4, CYP3A5 and CYP3A7 as well as CYP2C8, CYP2C9, CYP2C18 and CYP2C19 as well as CYP4F2, CYP4F3, CYP4F11, and CYP4F12) could be unambiguously differentiated from each other by MS, despite the fact that, for example, the sequence similarity of CYP2C9 compared to CYP2C19 is 91%. Table 4 shows an overview of the unique peptides used for specific isoform identification. Several unique tryptic peptides were identified for each CYP isoform except for CYP2C18 and CYP4F12 for which only one unique peptide was detected by LC-ESI-MS/ MS, although 15 and 18 unique peptides theoretically exist for CYP2C18 and CYP4F12, respectively (Table 2). On the other hand, CYP3A4 and CYP2C9 were identified with 14 and 12 unique tryptic peptides by LC-ESI-MS/MS, although only 7 and 6 unique tryptic peptides without missed cleavage sites occur (Table 2). Some peptides could not be used for unambiguous identification of protein isoforms with regular LC-ESI-MS/MS, although they are unique. This was the case for the CYP3A5 specific peptide with the sequence SAISLAEDEEWK which is quite similar to the CYP3A4 specific peptide SAISIAEDEEWK (Table 4). At sequence position 120, leucine is exchanged by isoleucine. Because of the same mass of leucine and isoleucine, the fragmentation pattern of both peptides in this approach is the same, and for this reason, it cannot be differentiated between CYP3A4 and CYP3A5 based on this peptide. In principle, it is possible to roughly estimate the relative amounts of the CYP isoforms in each sample by comparing the sequence coverage or the number of assigned peptides to the protein.44 Thus, our results might reflect the relative amounts of these CYPs in the samples (CYP3A4, CYP2C9 . CYP2C18, CYP4F12). Notably, hepatic expression of CYP2C18 protein is extremely low45 and CYP4F12 is reported to be mainly expressed in the gastrointestinal and urogenital epithelia.46 Additionally, relative semiquantification between the samples from different donors is possible, although this was not the aim of this study; thereby it can be determined that, for example, a higher amount of CYP2A6 is expressed in donor 3 and 4 (19 and 25 peptides assigned to the protein) in comparison to donor 1 or 2 (6 and 14 peptides assigned to the protein) (Table 2). However, CYP4F3 is an example that points out the limitation of this semiquantitative approach. The Journal of Proteome Research • Vol. 7, No. 11, 2008 4681

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Table 2. CYPs Identified within This Study in Microsomes from Donor 1-4 number of unique peptides assigned to the proteina

theoretical number of unique peptides in the protein sequencea, b

17 18 15 15 12 9 6 6 8 2 4 6 1

17 8 9 13 5 4 1 6 8 2 4 1 1

18 7 6 18 8 11 7 19 23 27 17 4 21

Donor 2 39% 35% 36% 27% 32% 33% 31% 31% 29% 17% 13% 12% 12% 5% 8%

17 14 14 14 12 14 11 14 11 8 5 6 6 2 3

5 14 14 3 8 8 4 2 9 1 1 1 1 2 3

7 18 17 11 6 11 8 4 18 7 14 18 13 25 23

1224 747 722 720 634 498 450 443 419 375 362 267 263 262 254 190 70 61

Donor 3 51% 52% 40% 27% 47% 25% 27% 38% 26% 29% 28% 16% 19% 17% 21% 15% 6% 2%

22 23 19 13 16 9 9 12 13 10 11 7 8 8 8 6 2 1

9 21 1 1 11 5 6 12 6 10 11 7 2 2 2 1 2 1

7 18 4 11 6 8 14 17 11 18 19 23 13 13 8 15 19 27

1395 1170 966 765 744 626 622 568 561 536 449 349 328 293 244 92

Donor 4 43% 53% 28% 36% 60% 30% 40% 34% 38% 35% 30% 19% 21% 26% 12% 8%

22 25 15 16 18 11 16 13 16 16 13 8 10 11 6 3

9 4 4 10 12 5 16 13 16 14 2 8 1 4 1 3

7 4 11 11 6 8 18 17 19 18 8 23 7 13 13 19

protein name

Swiss-Prot/Uniprot accession number (Version 54.4)

CYP1A2 CYP3A4 CYP2C9 CYP2E1 CYP4A11 CYP2C8 CYP4F3 CYP27A1 CYP8B1 CYP2J2 CYP2D6 CYP2A6 CYP7B1

P05177 P08684 P11712 P05181 P02928 P10632 Q08477 Q02318 Q9UNU6 P51589 P10635 P11509 O75881

981 957 493 435 391 255 190 176 174 147 136 134 44

Donor 1 44% 49% 38% 36% 32% 21% 12% 15% 19% 4% 16% 12% 2%

CYP3A4 CYP1A2 CYP2D6 CYP3A7 CYP2C9 CYP2C8 CYP4A11 CYP2A6 CYP2E1 CYP4F3 CYP3A5 CYP4F12 CYP4F11 CYP4V2 CYP8B1

P08684 P05177 P10635 P24462 P11712 P10632 P02928 P11509 P05181 Q08477 P20815 Q9HCS2 Q9HBI6 Q6ZWL3 Q9UNU6

944 908 796 657 554 514 448 435 326 314 255 233 211 113 99

CYP3A4 CYP2E1 CYP2A6 CYP3A7 CYP2C9 CYP4A11 CYP3A5 CYP2D6 CYP2C8 CYP1A2 CYP2B6 CYP8B1 CYP4F11 CYP2C19 CYP4F2 CYP2C18 CYP27A1 CYP2J2

P08684 P05181 P11509 P24462 P11712 P02928 P20815 P10635 P10632 P05177 P20813 Q9UNU6 Q9HBI6 P33261 P78329 P33260 Q02318 P51589

CYP3A4 CYP2A6 CYP3A7 CYP2C8 CYP2C9 CYP4A11 CYP1A2 CYP2D6 CYP2B6 CYP2E1 CYP4F2 CYP8B1 CYP4F3 CYP2C19 CYP4F11 CYP27A1

P08684 P11509 P24462 P10632 P11712 P02928 P05177 P10635 P20813 P05181 P78329 Q9UNU6 Q08477 P33261 Q9HBI6 Q02318

MASCOT protein score

sequence coverage

number of peptides assigned to the protein

a The number of identified unique peptides can be in some cases higher than the number of theoretical unique peptides in the sequence, because no peptides with missed cleavage sites were used to calculate the number of theoretical unique peptides. b Including peptides with a length of 5-20 amino acids and a mass between 800-4000 Da.

protein is identified with several but only 1 unique tryptic peptide in samples from donors 1, 2, and 4. This implicates that the nonidentification of CYP4F3 in donor 3 does not lead 4682

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to the assumption that the protein is absent in this sample. It may only have been that no unique peptide for this protein was recorded by MS/MS. Of course, for validation of such

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Identification of CYP Isoforms and Phosphorylation Sites by MS a

Table 3. List of Peptides Used for Single Peptide-Based Identifications protein name

Swis-Prot/Uniprot accession number (Version 54.4)

sequence

precursor mass (observed) [m/z]

precursor charge state (observed)

mass error (observed) [Da]

MASCOT peptide score

MASCOT expect value

Proteins Identified with One Unique Peptide CYP7B1 CYP2J2

O75881 P51589

R.LLFGIQYPDSDVLFR.Y

Donor 1 892.2

2+

0.36

44

0.025

R.VIGQGQQPSTAAR.E

Donor 3 657.0

2+

0.23

61

0.00039

Proteins Identified with Several but One Unique Peptide CYP4F3 CYP2A6

Q08477 P11509

K.WQLLASEGSAR.L R.GTGGANIDPTFFLSR.T

Donor 1 609.4 777

2+ 2+

0.17 0.27

58 91

0.00087 4.6 × 10-7

CYP4F3 CYP3A5 CYP4F12 CYP4F11

Q08477 P20815 Q9HCS2 Q9HBI6

K.VVLGLTLLR.F R.LGIPGPTPLPLLGNVLSYR.Q R.SITNASAAIAPK.D R.TLPTQGIDDFLK.N

Donor 2 492.4 989.3 572.4 674.5

2+ 2+ 2+ 2+

0.14 0.4 0.16 0.24

50 39 75 47

0.0045 0.096 1.7 × 10-5 0.011

CYP2A6 CYP3A7 CYP2C18

P11509 P24462 P33260

R.GTGGANIDPTFFLSR.T R.FGGLLLTEKPIVLK.A R.VPPLYQLCFIPV.-

Donor 3 777.1 510.1 723.5

2+ 3+ 2+

0.42 0.36 0.23

86 38 32

1.3 × 10-6 0.084 0.37

CYP4F3 CYP4F11

Q08477 Q9HBI6

K.VVLGLTLLR.F K.TLTQLVTTYPQGFK.L

Donor 4 492.4 799.1

2+ 2+

0.13 0.23

64 71

0.00016 4.7 × 10-5

a

All manually annotated MS/MS spectra can be found in supplementary material I in Supporting Information.

purposes, the measurements have to be repeated several times. Concerning quantitative CYP profiling, Jenkins et al. describe an interesting approach by using the ICAT reagent.47 LC-ESIMS/MS is used to relatively quantify the proteins afterward or MRM scans on a triple quadrupole instrument are performed for absolute quantification. Further, Lane et al. use 18O/16O labeling followed by LC-ESI-MS/MS for relative quantification of CYPs.48 Several efforts to discriminate between CYP isoforms by proteomicsandmassspectrometryaredescribedintheliterature.15,47-53 Most of the studies were performed with rat or mouse liver. But since the CYP pattern of these organisms is mainly different from that in human, studies are hardly comparable. Concerning P450 proteomics experiments performed with human tissue, Petushkova et al. show that the human CYP isoforms 1A2, 1B1, 2A6, 2C8, 2C9, 2C10, 2D6, 2E1, 3A4, 4A11, and 4F2 can be differentiated by 1D-SDS-PAGE followed by protein identification with MALDITOF-MS.49 Lane et al. describe the differentiation between the human CYP isoforms 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2A4, 4A11, 4F2, 4F11, 8B1, and 27A1 by 1D-SDS-PAGE and LC-ESI-MS/MS.48,53 In comparison to that, this is the first report that demonstrates the applicability of mass spectrometry to identify CYP2C18 and CYP3A7 on protein level. Both could only be measured on mRNA level in the past, including the already mentioned disadvantages of this approach, since no specific antibodies for these proteins are available. These results demonstrate the potential of MS to differentiate between very homologous CYP isoforms that exist in a highly complex mixture with large concentration differences and to estimate the relative amounts of each isoform in different samples. Further, this study presents a basis for the future development of fast and selective MS-based assays that especially can differentiate between the CYP isoforms 3A4, 3A5, 3A7,

and 2C8, 2C9, 2C18, 2C19. For this purpose, a comprehensive database of peptides unique for each CYP isoform and identifiable by MS can be used to identify appropriate reporter peptide ions for targeted MS approaches like multiple reaction monitoring (MRM)54 enabling fast and reliable CYP quantification.47 In combination with the stable isotope dilution techniques and internal standards,55 it is then possible to obtain absolute quantitative data for every CYP isoform. This could be the first step to unravel the role of the CYP isoforms 3A5, 3A7 and 2C18 in drug-metabolism. Identification of P450 Phosphorylation Sites. Next, we used the microsomal fractions to investigate the in vivo phosphorylation profiles of CYP proteins. We applied TiO2 based phosphopeptide enrichment followed by LC-ESI-MS/MS analysis for phosphopeptide sequencing. Microsomes were prepared with adequate precautions to prevent dephosphorylation of proteins by endogenous phosphatases. Using this approach, we detected a series of phosphopeptides from CYP proteins, including most of the important drug-metabolizing CYP isoforms (Table 1), namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6, CYP2E1, CYP3A4, CYP3A7, and CYP8B1 (Table 5). CYP1A2. This enzyme is important for metabolizing aromatic amines among others, like caffeine, for example.56 As already mentioned, CYP1A2 has been previously described to be phosphorylated by PKC,22 but the affected amino acid could not be localized so far. In this study, the CYP1A2 specific peptide IGpSTPVLVLSR was found to be phosphorylated at Ser82 (Table 5). The sequence was correlated to the respective spectrum obtained from donor 2 with a MASCOT score of up to 84 and an expect value down to 2.3 × 10-6 (the spectrum can be found in the supplemental material II in Supporting Information). The MS/MS data clearly assigned the phosphorylation site to Ser-82. Interestingly, NetPhos predicts a phosphorylation at Ser-89 and not at Ser-82, emphasizing the need Journal of Proteome Research • Vol. 7, No. 11, 2008 4683

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Table 4. Unique Tryptic Peptides Used for Unambiguous Identification of CYP3A/2C/4F Isoforms CYP3A subfamily

CYP2C subfamily

CYP3A4 LFPIAMR EVTNFLR GFCMFDMECHK SAISIAEDEEWKb GVVVMIPSYALHR LSLGGLLQPEKPVVLK LGIPGPTPLPFLGNILSYHK LSLGGLLQPEKPVVLKVESRa KLGIPGPTPLPFLGNILSYHKa VDFLQLMIDSQNSKETESHKa ETQIPLKLSLGGLLQPEKPVVLKa VWGFYDGQQPVLAITDPDMIK APPTYDTVLQMEYLDMVVNETLR SAISIAEDEEWKRa,b

CYP4F subfamily

CYP2C8 ICAGEGLAR VQEEIDHVIGR EALIDNGEEFSGR YSDLVPTGVPHAVTTDTK YGLLLLLKHPEVTAKa VKEHQASLDVNNPRa SVDDLKNLNTTAVTKa RICAGEGLARa KLPPGPTPLPIIGNMLQIDVKa SDYFMPFSAGKRa YSDLVPTGVPHAVTTDTKFRa

CYP4F2 SVINASAAIAPK HVTQDIVLPDGR VWMGPISPLLSLCHPDIIR CYP4F3 VVLGLTLLR WQLLASEGSAR CYP4F11 ILPTHTEPR TLPTQGIDDFLK TLTQLVTTYPQGFK

CYP2C9 GIFPLAER YFMPFSAGK SHMPYTDAVVHEVQR YIDLLPTSLPHAVTCDIK LPPGPTPLPVIGNILQIGIK GKLPPGPTPLPVIGNILQIGIKa VKEHQESMDMNNPQDFIDCFLM(Ox)Ka ILSSPWIQICNNFSPIIDYFPGTHNK HNQPSEFTIESLENTAVDLFGAGTETTSTTLR EKHNQPSEFTIESLENTAVDLFGAGTETTSTTLRa EHQESMDMNNPQDFIDCFLMK LPPGPTPLPVIGNILQIGIKDISKa

CYP3A5 LDTQGLLQPEKPIVLK DSIDPYIYTPFGTGPR LGIPGPTPLPLLGNVLSYR LKEMFPIIAQYGDVLVRa SAISLAEDEEWKb SAISLAEDEEWKR,b ETQIPLKLDTQGLLQPEKPIVLKa CYP3A7 FALVNMK FNPLDPFVLSIK FGGLLLTEKPIVLK KVISFLTKa

CYP4F12 SITNASAAIAPK

CYP2C18 VPPLYQLCFIPV CYP2C19 GHFPLAER HFLDEGGNFK NLAFMESDILEK VKEHQESMDINNPRa

a

One missed cleavage site.

b

Peptides hardly distinguishable with this approach.

Table 5. Overview of Identified CYP Phosphorylation Sitesa

phosphorylated CYP isoform

peptide sequence

CYP1A2 CYP2A6 CYP2A6 CYP2B6 CYP2C8 CYP2D6 CYP2E1e CYP3A4 CYP3A7 CYP8B1

IGpSTPVLVLSR RFpSIATLRb NYpTM(Ox)SFLPRc RFpSVTTM(Ox)R RFpSLTTLRd RFpSVSTLR RFpSLTTLRd SLLpSPTFTSGKf SLLpSPTFTSGKf SVQGDHEMIHSApSTK

identified NetPhos phosphorylation phosphorylation site prediction scoreh Ser-82 Ser-131 Thr-488 Ser-128 Ser-127 Ser-135 Ser-129 Ser-134 Ser-134 Ser-127

0.03 0.95 0.03 0.99 0.99 0.99 0.99 0.93 0.93 0.99

described in literature as phosphorylated not site-specificI Ser-129j Thr-264, Ser-420g, k Ser-420g,k -

precursor detected in mass MASCOT MASCOT donor (bold: given (observed) Charge peptide expect MASCOT values) [m/z] state score value 1,2, 4 1,2,3,4 4 4 1,2,3,4 3, 4 1,2,3,4 4 4 1, 2

611.4 522.3 612.9 547.3 537.4 523.3 537.4 609.4 609.4 569.6

2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+

84 49 37 29 44 54 44 49 49 44

2.3 × 10-6 0.0072 0.12 0.68 0.017 0.0023 0.017 0.0077 0.0077 0.031

a All manually annotated MS/MS spectra of the identified phosphopeptides can be found in the supplementary material II in Supporting Information. Phosphopeptide homologue in CYP2A13. c Phosphopeptide homologue in CYP2A7 and CYP2A13. d Phosphopeptide homologue in CYP2C8 and CYP2E1. e Two other not localized phosphorylation sites in the phosphopeptide: HSAERLYTMDGITVTVADLFFAGTETTSTTLR. f Phosphopeptide homologue in CYP3A4, CYP3A5 and CYP3A7. g Phosphopeptide FLPERFpSK homologue in CYP3A4 and CYP3A7 h Referred to refs 41 and 42. I Referred to ref 22. j Referred to ref 20. k Referred to ref 21. b

to obtain high-quality experimental data rather than relying on current computational predictions for phosphorylation site assignments. CYP2A6. CYP2A6 is responsible for the hydroxylation of certain aromatic compounds such as coumarin and nicotine.56 Two novel phosphorylation sites were located in the tryptic peptides RFpSIATLR (Ser-131) and NYpTM(Ox)SFLPR (Thr-488) (Table 5). It has to be mentioned that RFpSIATLR is homologue 4684

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in CYP2A13 and NYpTM(Ox)SFLPR is homologue in CYP2A13 and CYP2A7, although both isoforms were not unambiguously identified in any donor sample. The sequence RFpSIATLR was matched to the accordant spectrum acquired from donor 4 with a MASCOT score of up to 49 and an expect value down to 0.0072. NYpTM(Ox)SFLPR only identified in donor 4 was correlated to the accordant spectrum with a MASCOT score of 37 and an expect value of 0.12 (the spectra can be found in

Identification of CYP Isoforms and Phosphorylation Sites by MS

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Figure 3. Annotated MS/MS-spectrum of the phosphorylated CYP2D6 specific peptide with the sequence RFpSVSTLR and a MASCOT score of 54 with an accordant expect value of 0.0023. Manual interpretation of the spectrum unequivocally identified Ser-135 to be phosphorylated.

the supplemental material II in Supporting Information). NetPhos predicts the Ser-131 phosphorylation site with a score of 0.95, but does not predict Thr-488 to be phosphorylated. CYP2B6. The role of this enzyme in xenobiotic metabolism has been underestimated in the past, but several important drugs like Efaviranz and Cyclophosphamide were recently reported to be substrates.5 Our data show that liver CYP2B6 is phosphorylated at Ser-128 (Table 5). This is confirmed by a NetPhos value of 0.99. The relevant spectrum obtained from donor 4 was correlated to the unique CYP2B6 sequence RFpSVTTM(Ox)R with a MASCOT score of 29 and an expected value of 0.68. Manual validation confirmed the correctness of the findings (the spectrum can be found in the supplemental material II in Supporting Information). Interestingly, the rat ortholog CYP2B1 was also described to be phosphorylated at Ser-128.22,57 The effect of Ser-128 phosphorylation on CYP2B1 may be twofold in that both enzyme deactivation as well as decreased mitochondrial import rate have been described.18,57 CYP2E1 and CYP2C8. CYP2E1, the only member of the human CYP2E subfamily, metabolizes small molecules with hydrogen bond acceptors like acetone.3 A PKA sequence recognition motif has already been described to be phosphorylated at Ser-129 and the degree of phosphorylation could be correlated to the metabolic activity of CYP2E1.20 CYP2C8 metabolizes weak acidic compounds with a hydrogen bond acceptor as for example the anticancer drug Taxol3 and has not previously been reported as phosphorylated. In our study, we could however distinguish between CYP2E1 and CYP2C8, but we identified the tryptic peptide with the sequence RFpSLTTLR to be phosphorylated, that is homologue in CYP2C8 and CYP2E1. The respective phosphopeptide spectrum recorded from donor 4 was matched to the sequence with a MASCOT score of up to 44 and an expect value down to 0.017 (the spectrum can be found in the supplemental material II in Supporting Information). The appropriate phosphorylated

amino acid would be Ser-129 in CYP2E1 and Ser-127 in CYP2C8 for which the NetPhos prediction value is 0.99 in both cases. CYP2C8 and CYP2E1 as well as the phosphopeptide could be identified in all four donor samples. This means we cannot differentiate between the phosphorylation of these two isoforms. Furthermore, the CYP2E1 specific peptide HSAERLYTMDGITVTVADLFFAGTETTSTTLR was also found by mass spectrometry to have one or two phosphorylation sites. However, the MS/MS spectrum did not allow unambiguous localization of the phosphorylation sites. Indeed, Ser-279 (underlined) was predicted by NetPhos to be phosphorylated with a probability value of 0.995. CYP2D6. Many different xenobiotics containing basic nitrogen atoms like Dextromethorphan are metabolized by the highly polymorphic and very important CYP2D6.5 In this study, Ser-135 was found to be phosphorylated. To our knowledge, there has been no previous report on possible phosphorylation of this enzyme. The accordant spectrum acquired from donor 3 was aligned to the unique tryptic sequence RFpSVSTLR with a MASCOT score of up to 54 and an expect value down to 0.0023 (Figure 3). NetPhos predicts a value of 0.997 for Ser135 phosphorylation. CYP3A4/5/7. CYP3A4 is the major form of human CYPs both with respect to the amount as well as the importance in drug metabolism. The enzyme substrates are large and diverse in structure. About 50% of all available drugs are metabolized to some degree by this subfamily.4 CYP3A4 has already been described to become phosphorylated at Thr-264 and Ser-420 by PKC treatment,21 although the functional effect remained ambiguous. We could not confirm those phosphorylation sites in vivo but localized a new phosphorylation site in donor 4 at Ser-134 included in the sequence of SLLpSPTFTSGK. The corresponding spectrum was annotated with a MASCOT score of 49 and an expect value of 0.0077 (the spectrum can be found in the supplemental material II in Supporting Information). Journal of Proteome Research • Vol. 7, No. 11, 2008 4685

research articles NetPhos predicts a value of 0.93 for Ser-134 phosphorylation. The identified tryptic peptide is identical to the closely related CYP3A isoforms CYP3A5 and CYP3A7. However, Ser-134 phosphorylation was identified in a donor with no detectable CYP3A5. So it is most likely that the phosphorylated peptide was derived from CYP3A4 and/or CYP3A7. However, it cannot be distinguished between the phosphorylation of both isoforms. CYP8B1. This isozyme metabolizes mainly sterols and is important for bile acid biosynthesis.3 It was found to be phosphorylated at Ser-127. The accordant spectrum obtained from donor 2 was correlated to the unique tryptic sequence SVQGDHEMIHSApSTK with a MASCOT score of up to 44 and an expect value down to 0.031 (the spectrum can be found in the supplemental material II in Supporting Information). NetPhos predicts a value of 0.987 for Ser-127 phosphorylation.

Conclusions CYPs within one subfamily generally exhibit high sequence similarity, so that immunoblotting-based methods often fail to reliably distinguish them. This is particularly the case for the members of subfamilies CYP2C and CYP3A which are only partially distinguishable by commercially available antibodies, but are very important to analyze because of their drugmetabolizing function. We detected and distinguished the highly similar isoforms CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12 in human liver microsomes. As shown here, mass spectrometry provides a sensitive and specific alternative to the classical approaches for isoform-specific CYP detection and characterization in complex human tissue fractions. Using affinity enrichment and mass spectrometry, we detected and localized eight in vivo CYP phosphorylation sites, seven of which were novel. This is to our knowledge the first report on the phosphorylation of the human CYP2A6, CYP2B6, CYP2D6 and CYP8B1 isoforms. On the basis of these findings, we expect that most of the drug-metabolizing P450 enzymes, especially those that metabolize the majority of xenobiotica, are phosphorylated to some degree. A current limitation of this study is that no discrimination between the phosphorylation of CYP2E1 and CYP2C8 as well as CYP3A4 and CYP3A7 was possible. The use of other proteases for digestion of CYPs prior to MS analysis may result in the generation and detection of unique and distinct phosphorylated peptides for these isoforms. Our data now serve as a basis for further analysis of CYPs and their phosphorylation. We are currently establishing quantitative mass spectrometry methods to further investigate the molecular composition and phosphorylation patterns of CYP enzymes in order to elucidate the functional consequences for enzyme activity, intracellular transport, and degradation processes. Abbreviations: ER, endoplasmic reticulum; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; TFA, triflouroacetic acid; ACN, acetonitrile; FA, formic acid; LC, liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; ESI, electrospray ionization; i.d., inner diameter; amu, atomic mass unit; RP, reversed-phase; PAGE, polyacrylamide gel electrophoresis; EDTA, ethylene diamine tetraacetic acid; PKC, protein kinase C; TOF, time-of-flight; ICAT, isotopecoded affinity tag; MRM, multiple reaction monitoring.

Supporting Information Available: Manually annotated MS/MS spectra of the identified peptides used for single -peptide based protein identifications (supplimental 4686

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Redlich et al. material I) and of identified phosphopeptides (supplemetal material II). This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgment. Gorden Redlich was a 3-month visiting research fellow in the Protein Research Group at the University of Southern Denmark and was supported by the German Academic Exchange Service (DAAD exchange scholarship). This work was also funded by the HepatoSys network project of the German Ministry of Education and Science (BMBF, Hepatosys, grant 0313080J and 0313080I). Ole N. Jensen is a Lundbeck Foundation Research Professor. References (1) Aguiar, M.; Masse, R.; Gibbs, B. F. Regulation of cytochrome P450 by posttranslational modification. Drug Metab. Rev. 2005, 37 (2), 379–404. (2) Omura, T. Mitochondrial P450s. Chem.-Biol. Interact. 2006, 163 (1-2), 86–93. (3) Lewis, D. F. 57 varieties: the human cytochromes P450. Pharmacogenomics 2004, 5 (3), 305–18. (4) Ingelman-Sundberg, M. Human drug metabolising cytochrome P450 enzymes: properties and polymorphisms. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2004, 369 (1), 89–104. (5) Zanger, U. M.; Klein, K.; Saussele, T.; Blievernicht, J.; Hofmann, M. H.; Schwab, M. Polymorphic CYP2B6: molecular mechanisms and emerging clinical significance. Pharmacogenomics 2007, 8 (7), 743–59. (6) Rostami-Hodjegan, A.; Tucker, G. T. Simulation and prediction of in vivo drug metabolism in human populations from in vitro data. Nat. Rev. Drug Discovery 2007, 6 (2), 140–8. (7) Ekins, S.; reyev, S.; Ryabov, A.; Kirillov, E.; Rakhmatulin, E. A.; Bugrim, A.; Nikolskaya, T. Computational prediction of human drug metabolism. Expert Opin. Drug Metab. Toxicol. 2005, 1 (2), 303–24. (8) Shimada, T.; Yamazaki, H.; Mimura, M.; Inui, Y.; Guengerich, F. P. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 1994, 270 (1), 414–23. (9) Gelboin, H. V.; Krausz, K. Monoclonal antibodies and multifunctional cytochrome P450: drug metabolism as paradigm. J. Clin. Pharmacol. 2006, 46 (3), 353–72. (10) Boobis, A. R.; Edwards, R. J.; Adams, D. A.; Davies, D. S. Dissecting the function of cytochrome P450. Br. J. Clin. Pharmacol. 1996, 42 (1), 81–9. (11) Kuehl, P.; Zhang, J.; Lin, Y.; Lamba, J.; Assem, M.; Schuetz, J.; Watkins, P. B.; Daly, A.; Wrighton, S. A.; Hall, S. D.; Maurel, P.; Relling, M.; Brimer, C.; Yasuda, K.; Venkataramanan, R.; Strom, S.; Thummel, K.; Boguski, M. S.; Schuetz, E. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat. Genet. 2001, 27 (4), 383– 91. (12) Hustert, E.; Haberl, M.; Burk, O.; Wolbold, R.; He, Y. Q.; Klein, K.; Nuessler, A. C.; Neuhaus, P.; Klattig, J.; Eiselt, R.; Koch, I.; Zibat, A.; Brockmoller, J.; Halpert, J. R.; Zanger, U. M.; Wojnowski, L. The genetic determinants of the CYP3A5 polymorphism. Pharmacogenetics 2001, 11 (9), 773–9. (13) Westlind-Johnsson, A.; Malmebo, S.; Johansson, A.; Otter, C.; ersson, T. B.; Johansson, I.; Edwards, R. J.; Boobis, A. R.; IngelmanSundberg, M. Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab. Dispos. 2003, 31 (6), 755–61. (14) Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature 2003, 422 (6928), 198–207. (15) Wang, Y.; Al.Gazzar, A.; Seibert, C.; Sharif, A.; Lane, C.; Griffiths, W. J. Proteomic analysis of cytochromes P450: a mass spectrometry approach. Biochem. Soc. Trans. 2006, 34 (Pt 6), 1246–51. (16) Jensen, O. N. Interpreting the protein language using proteomics. Nat. Rev. Mol. Cell. Biol. 2006, 7 (6), 391–403. (17) Oesch-Bartlomowicz, B.; Oesch, F. Cytochrome.P450 phosphorylation as a functional switch. Arch. Biochem. Biophys. 2003, 409 (1), 228–34. (18) Anandatheerthavarada, H. K.; Biswas, G.; Mullick, J.; Sepuri, N. B.; Otvos, L.; Pain, D.; Avadhani, N. G. Dual targeting of cytochrome P4502B1 to endoplasmic reticulum and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at ser128. EMBO J. 1999, 18 (20), 5494–504.

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