A Targeted Proteomics Approach to the Identification of Peptides

Chem. Res. Toxicol. 2009, 22, 853–862

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A Targeted Proteomics Approach to the Identification of Peptides Modified by Reactive Metabolites Manuel Tzouros and Axel Pa¨hler* Drug Metabolism and Pharmacokinetics, Non-Clinical Safety, Pharmaceuticals DiVision, F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland ReceiVed NoVember 11, 2008

Covalent binding of reactive metabolites is generally accepted as one underlying mechanism of druginduced toxicity. However, identification of protein targets by reactive metabolites still remains a challenge due to their low abundance. Here, we report the development of a highly selective proteomics workflow for the targeted identification of peptides modified by reactive metabolites. An equimolar mixture of non- and radiolabeled furan containing 2-amino-pyrimidine drug candidate (1 and 14C1-1) along with rat liver microsomes were used for the in vitro generation of reactive metabolites. Liver microsomal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, modified protein bands were highlighted by autoradiography and in-gel digested, and peptides were fractionated by strong cation exchange chromatography. Fractions enriched in modified peptides, as determined by radioactivity levels, were subjected to nanoLC-MS/MS and unambiguously detected based on their unique 12C/14C MS isotope pattern fingerprint. The peptide detection step could be automated using isotope pattern recognition software. Peptide sequencing, identification of the site of modification, and reactive metabolite characterization were achieved by MS2 and MS3 experiments using high-resolution and accurate mass detection. This approach led to the identification of four modified peptides originating from three drugmetabolizing enzymes, MGST1, FMO1, and P450 2C11. These revealed modifications by five different metabolite structures. This approach is generally suitable for the identification and characterization of modified proteins and metabolite structures involved in covalent binding and may serve as a valuable tool to link protein targets with clinically relevant toxicities. Introduction Bioactivation of pharmaceuticals to reactive metabolites is an unwanted drug property that has been demonstrated to lead to adverse drug reactions, liver toxicity, and market withdrawal (1-5). These highly reactive electrophilic species are the oxidation products formed by drug-metabolizing enzymes and can further irreversibly react with cellular macromolecules such as proteins by formation of a covalent bond with a nucleophilic residue (6). In most pharmaceutical companies, undesired metabolic activation is usually minimized during the lead optimization process in drug discovery to avoid excessive protein covalent binding (Cvb)1 (7, 8). Typically, quantification of Cvb is assessed in vitro by incubating radiolabeled drug with liver microsomal preparations, where nonextractable radioactivity associated with proteins is measured. Cvb to hepatic proteins serves as a surrogate marker for potential toxicity risks induced by excessive reactive metabolite formation. In addition, the structure of the reactive metabolites is probed by using reactive intermediate trapping experiments (9-11). However, despite being of paramount importance, these trapping assays do not address some important issues that would allow a better understanding of the cascade of events leading to Cvb. First, * To whom correspondence should be addressed. Tel: +41616889920. Fax: +41616882908. E-mail: [email protected]. 1 Abbreviations: ADME, adsorption, distribution, metabolism, and excretion; CID, collision-induced dissociation; CNS, central nervous system; CPM, counts per minute; Cvb, covalent binding; dAla, dehydro-alanine; ∆m/z, difference in m/z; ∆RA, difference in relative abundance; FMO1, dimethylaniline monooxygenase (N-oxide-forming) 1; HLM, human liver microsomes; MGST1, microsomal glutathione S-transferase 1; RLM, rat liver microsomes; SCX, strong cation exchange.

these assays give no information about which protein is targeted by the reactive metabolite(s). Second, the structure of the metabolite bound to a trapping agent may not necessarily reflect the one attached to a protein. Third, very reactive species may not be detected at all if they react immediately with residues present at the enzyme’s active site; therefore, the intermediate is precluded from diffusing and meeting trapping agents. This knowledge about target proteins modified by reactive metabolites is of utmost importance to further develop our understanding on the link between Cvb and drug-induced toxicity. Large-scale protein identifications from complex mixtures are routinely performed using “shotgun” approaches, combining gel electrophoresis [one-dimensional (1D) and two-dimensional (2D) SDS-PAGE], enzymatic digestion, and nanoscale liquid chromatography coupled to mass spectrometry (LC-MS/MS) (12-14). As a consequence to these recent improvements in the field of proteomics, analysis of protein targets of reactive drug metabolites has also become a more accessible task (7, 15). In that particular case, to locate modified proteins that have to be considered for further identification within a complex mixture, experiments are often performed with radiolabeled (14C or 3H) substrate and targets visualized by a radioactivity imaging technique (16). Using 2D SDS-PAGE, autoradiography, and MS detection, a total of 23 protein targets of acetaminophen could be identified from a rat liver homogenate (17). However, no spectral evidence for a modified peptide was reported, still leaving a doubt about the veracity of the proposed target. Since this pioneering work, very few examples of successful identification by MS of peptides modified by reactive drug metabolites and obtained by bioactivation have been reported in the literature

10.1021/tx800426x CCC: $40.75  2009 American Chemical Society Published on Web 03/25/2009

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Scheme 1. Targeted Proteomics Workflow for Sample Preparation and Analysis of Microsomal Proteins Modified by Reactive Drug Metabolites Using a 1:1 Mixture of Nonand Radiolabeled Substrate

Tzouros and Pähler

pattern. This characteristic signature is readily observed by MS and allows distinction between modified and unchanged peptides. This step is performed in an automated way using an isotope pattern recognition software. Peptide sequencing by MS/ MS combined with database searches finally reveals the identities of the protein targets, the sites of modification, and the reactive metabolites.

Experimental Procedures

(18-21). In all of these cases, the presence of a modified peptide could be testified, but direct sequencing by MS failed. This might be due to the low abundance of the adducted peptides and to facile loss of the attached metabolite caused by hydrolysis. Recently, two reports on raloxifene-modified P450 3A4 successfully identified a cysteine and a tyrosine as the modified amino acid residues by LC-MS/MS (22, 23). Furthermore, 17-R-ethynylestradiol was shown to modify Ser360 in both P450 2B1 and 2B6 (24). In addition, an elegant method using model biotinylated reactive electrophiles was successfully applied to identify modified cysteine residues of proteins originating from human liver microsomes (HLMs) (25). However, no generally applicable method exists to date that allows one to unequivocally designate which protein was modified by reactive drug metabolites and where the modifications are located on the protein. This information is mandatory to relate protein modification by reactive metabolites to certain types of adverse events. In this paper, we describe a novel targeted proteomics approach to detect, enrich, and identify peptides modified by reactive drug metabolites derived from rat liver microsomal proteins. The analytical strategy is based on the use of an equimolar mixture of non- and radioisotope-labeled (14C1) substrate for the in vitro incubations. The protein mixture is resolved by 1D SDS-PAGE, modified protein bands visualized by gel autoradiography, and selected for in-gel digestion using trypsin (Scheme 1). Peptides are then fractionated by strong cation exchange (SCX) chromatography and radioactivity containing fractions analyzed by nanoLC-MS/MS. More than a mean to track modified proteins and peptides, the non- and radiolabeled substrate mixture strategy results in the presence of modified peptides that exhibit a unique 12C/14C isotopic

Chemicals and Reagents. 2-Amino-4-furan-2-yl-6-(2-pyridin2-ylethoxy)pyrimidine-5-carbonitrile (1) and its radiolabeled analogue 14C1-1 (specific activity, 52.6 µCi/µmol; 99.8% radiochemical purity; see Figure 1 for the structure and position of 14C-atom) were synthesized in-house (Hoffmann-La Roche Ltd., Basel, Switzerland). Proteomics grade trypsin, NADPH, and iodoacetamide were purchased from Sigma-Aldrich (Buchs, Switzerland). DTT was obtained from AppliChem (Darmstadt, Germany). Male rat liver microsomes (RLMs) (Wistar Han) were purchased from BD Gentest (Allschwil, Switzerland). HPLC grade water and acetonitrile were purchased from Merck (Darmstadt, Germany). Microsomal Incubations. A 1:1 mixture of 1 and 14C1-1 (final total concentration of 10 µM) was incubated at 37 °C with liver microsomes (1 mg/mL of protein) and NADPH (1 mM) in sodium phosphate buffer (100 mM, pH 7.4) for 30 min. The total incubation volume was 2 mL. The incubation reactions were initiated by the addition of NADPH after a 3 min preincubation and were stopped by placing the tubes on ice. Control incubations were conducted lacking either NADPH or substrate. The samples were desalted by gel filtration using Zeba desalting spin columns (Pierce, Rockford, IL) equilibrated with Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, and 2% SDS) according to the manufacturer’s procedure. This step allowed us to remove >98% of the total radioactivity, that is, excess of unbound substrate (data not shown). SDS-PAGE and Autoradiography. Proteins were denatured by the addition of DTT (27 mg/mL) and bromophenol blue (traces) and boiling at 95 °C for 5 min. Proteins (25 µg for analytical and 2 mg for preparative separations) were resolved on a 12% SDSPAGE using Tris-glycine SDS running buffer, and gels were stained with Coomassie blue (Invitrogen, Carlsbad, CA). For autoradiography, gels were dried and exposed for 5-7 days to BAS-III imaging plates and visualized using a FLA-3000 scanner (FujiFilm, Tokyo, Japan). In-Gel Tryptic Digestion and Peptide Extraction. Radioactive bands were cut out of the preparative gels, diced into ∼1 mm3 pieces, and placed in two separate 15 mL conical tubes (Becton Dickinson, Franklin Lakes, NJ). Gel pieces were washed and shrinked three times with ammonium bicarbonate (50 mM) and acetonitrile, respectively, to remove the staining. Proteins were then reduced with DTT (6.5 mM) and heating at 50 °C for 45 min and alkylated with iodoacetamide (54 mM) at room temperature for 1 h in the dark. After two additional washing and shrinking steps, tubes were placed on ice, and trypsin solution (2.5 ng/µL) was added, kept on ice during reswelling of the gel pieces, and finally incubated at 37 °C overnight. If necessary, additional ammonium bicarbonate was added after 1 h of incubation, to ensure that the gel pieces were completely covered by buffer. Peptides were extracted with 50% acetonitrile, and samples were dried in a vacuum concentrator. It is noteworthy that no acid was added to the extraction solvent to minimize the risks of hydrolysis of the bound metabolites from the peptides. SCX. SCX chromatography was performed using a Zorbax BioSCX-Series II column (0.8 mm i.d. × 50 mm length, 3.5 µm), a HTS PAL autosampler equipped with a fraction collection system (CTC Analytics, Zwingen, Switzerland), a Shimadzu SCL-10Avp controller, a LC-10ADvp binary pump, and a SPD-10Avp UVdetector (Shimadzu, Tokyo, Japan). Flow was passively split (ratio 10:1) prior to the injection loop to a final rate of 50 µL/min. Before fractionation by SCX, protein digests were desalted by StageTips using a small plug of C18 material (3 M Empore C18 extraction disk) placed into a GELoader tip (Eppendorf) similar to what has

Modified Peptides by ReactiVe Metabolites

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Figure 1. Furan-containing 2-amino-pyrimidine derivatives 1 and 14C1-1 used as model substrate for method optimization and proof of concept and tentative metabolic transformations leading to GSH and protein adducts observed in liver microsomes.

been previously described (26) onto which ∼5 µL of Aqua C18 (5 µm particle size, 200 Å) material (Phenomenex, Torrance, CA) was placed. The eluate was dried completely and subsequently reconstituted in 20% acetonitrile and 0.05% formic acid. After injection, a linear gradient of 1%/min solvent B (500 mM KCl in 20% acetonitrile and 0.05% formic acid, pH 3.0) was performed. Two successive chromatographic runs were carried out; fractions were collected either in a LumaPlate-96 and radioactivity was detected using a TopCount NXT (PerkinElmer, Boston, MA) or in a 96 well plate for further nanoLC-MS/MS analysis. LC-MS. Nanoflow LC-MS/MS was performed by coupling an Agilent 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) to an LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany). A home-built vented column system, consisting of a 10 mm Aqua C18 trapping column (packed in-house, 100 µm i.d.) and a 161 mm Reprosil-Pur C18-AQ (Dr. Maisch GmbH, Ammerbuch, Germany) analytical column (packed in-house, 75 µm i.d., 3 µm particle size, 120 Å) was used for online desalting and separation of the peptides. The flow rate was passively split from 0.3 mL/min to ∼200 nL/min. Trapping of the peptides was performed at 5 µL/min for 10 min with 100% solvent A (0.6% acetic acid), and elution was carried out using the following gradient of solvent B (80% acetonitrile and 0.6% acetic acid): 0-50% B in 35 min, 50-100% B in 3 min, and 100% B for 2 min. The eluent was electrosprayed via a distal-coated emitter tip (New Objective, Cambridge, MA), butt-connected to the analytical column. Between the high voltage supply and the needle, an additional 70 MΩ resistor was placed to reduce ion current. Data-Dependent LC-MS/MS. The protein digests from the gel bands and the individual SCX fractions of interest were analyzed by a generic MS/MS setting to allow for the identification of modified peptides. The MS method used was as follows: The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS. Survey full scan MS spectra were acquired from m/z 300 to m/z 1500 in the Orbitrap with a resolution of R ) 100000 at m/z 400 after accumulation to a target value of 500000 in the linear ion trap for a maximum time of 250 ms and with a microscan count of 1. The detected ions were recalibrated on the fly using ambient air polysiloxanes as lock masses (27). The four most intense ions at a threshold above 1000 were selected for collision-induced dissociation (CID) in the linear ion trap at a normalized collision energy of 35% after accumulation to a target value of 10000 for a maximum time of 500 ms and with an isolation width of 2.5 amu. The nonpeptide monoisotopic recognition was enabled for the data-dependent algorithm to select the 12C peak as the parent monoisotopic m/z. High-Resolution LC-MS/MS. The SCX fractions containing peptide adducts (presenting a characteristic 12C/14C isotopic pattern, see below) were additionally reanalyzed to obtain high-resolution

MS/MS for detailed characterization of peptide adducts. The method used was as follows: Ions of interest were continuously fragmented by CID in the linear ion trap and were analyzed in the Orbitrap with a resolution of R ) 7500, after accumulation to a target value of 100000 for a maximum time of 1 s and with an isolation width of 4.5 amu. When needed, fragment ions of interest were further analyzed by MS3. Alternatively, to obtain information about the occurrence of immonium ions, the same was performed using higher energy collisional dissociation (HCD) at a normalized collision energy of 35% (28). Data Analysis. Mass chromatograms from the data-dependent runs were searched for ions displaying a characteristic 12C/14C isotopic pattern using MetWorks 1.1.0 (Thermo Fisher Scientific). Taking five isotopic peaks into consideration for the data mining, theoretical patterns were set at ∆m/z of 0.334, 0.668, 1.002, and 1.336 for triply charged ions and ∆m/z of 0.501, 1.002, 1.503, and 2.004 for doubly charged ions, with differences in relative abundances (∆RAs) of 0.9, 1.2, 0.9, and 0.5, respectively, for both charge states, with respect to the 12C-monoisotopic peak of the unlabeled peptides. The matching tolerance was set at 0.5%. The peaks identified using MetWorks were further manually validated. Modified peptide MS/MS data were manually interpreted using the high-resolution data, and parent proteins were identified by performing sequence pattern searches using fuzzpro from the EMBOSS (29) and the Swiss-Prot/TrEMBL database restricted to the rat organism (release 56.1 of September 2, 2008, 7212 entries). The structure of the attached metabolite was then deduced from the difference between the measured peptide mass and the calculated mass for the unmodified peptide. Furthermore, to confirm the presence of the modified proteins, raw data from the data-dependent runs were processed with Proteome Discoverer 1.0 (Thermo Fisher Scientific), and searches were done using SEQUEST and the abovementioned database allowing two enzyme missed cleavages, carbamidomethylation (Cys) as a static and oxidation (Met), deamidation (Asn, Gln) as differential modifications, respectively. The peptide tolerance was set to 10 ppm, the fragment ion tolerance was set to 1.0 amu, and the search results were filtered by applying a XCorr threshold of 1.8.

Results Analytical Strategy. Radioisotope-labeled substrates are widely used in drug metabolism studies to trace and quantify metabolites as well as protein Cvb (8). In this study, radioactivity was used to monitor the fate of the modified proteins or peptides during the whole sample preparation procedure preceding LCMS analysis. In addition, by using an equimolar ratio of nonand radiolabeled substrate 1 and 14C1-1 for the experiments, any

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modified peptide displayed a characteristic isotopic doublet with a mass difference of 2 Da in the MS that clearly allowed distinction from a whole pool of unmodified peptides. Furthermore, it aided in the localization of the modified amino acid residue during structure elucidation of the modified peptides, since the 12C/14C isotopic doublet was also observed in the MS/ MS for certain fragment ions. The furan-containing 2-amino-pyrimidine derivative 1 and its radiolabeled analogue 14C1-1 (Figure 1) were used as model substrates in these studies since this screening hit from a central nervous system (CNS) indication was known to form adducts with GSH and to cause high protein Cvb. Compound 1 causes strong time-dependent inactivation of cytochrome P450 3A4 in HLMs, similar to what has been reported for an analogous chemical series of 1 (30). Reactive metabolite formation and protein Cvb of 1 most likely involve the furan moiety present in the molecule that is known to be activated by cytochrome P450-mediated oxidation to highly reactive furan epoxide or its isomeric γ-ketoenal form (as depicted in Figure 1) (31-33). The presence of this type of reactive intermediates further supports that the expected amino acid to be alkylated should be a cysteine rather than a lysine or a histidine (34). In addition, this model substrate is an excellent candidate for the SCX enrichment strategy (see below), since 1 possesses two basic groups, a pyridinyl and a 2-amino-pyrimidine moiety. However, on the basis of the pKa values for the two groups and the typical pH operated during SCX, only the pyridinyl group should act as proton acceptors. The initial system investigated for a proof of concept was RLMs because 1 exhibited high turnover and consequently high Cvb values in this in vitro system. PAGE of Modified Proteins and Tryptic Digestion. Following in vitro generation of reactive metabolites, liver microsomal proteins were desalted by gel filtration chromatography, and a 1% fraction of the overall sample was resolved by SDS-PAGE to visualize modified proteins by autoradiography (Scheme 1). The gel filtration step not only allowed desalting and exchanging of the buffer necessary for the following electrophoresis, it also removed the excess substrate. The latter was particularly helpful, since remaining radioactive substrate in the incubation would cause higher background noise in the gel autoradiography, making recognition of the modified proteins more difficult. The autoradiogram revealed three major bands (named 1-3 in Figure 2) associated with significant radioactivity levels: Two bands, 1 and 2, were located in the 40-60 kDa region, and a strong band 3 was observed in the low molecular mass region around 16 kDa (Figure 2). It is notable that the modified bands 1 and 2 are positioned in the molecular mass region of the gel where the P450 oxidoreductases are expected to migrate (35). In addition to these three intense bands, other minor bands located in the 55-98 kDa and in the 22-36 kDa regions evidenced modified proteins but were not further identified. Control incubations, lacking either NADPH or substrate, did not reveal any modified protein bands. A larger scale preparative separation was performed with the remaining incubate (ca. 2 mg of protein) to be used for the consecutive analytical steps (see Scheme 1). After coomassie staining, the regions of the large gel known to contain radioactivity were cutout, and proteins were in-gel reduced, alkylated, and further digested with trypsin. The final extraction of the peptides from the gel pieces was performed without addition of any acid to avoid hydrolysis and removal of the metabolites attached to the peptides. Hydrolysis of modified peptides when treated with organic acid is one possible reason why their mass spectrometric detection often failed (19, 20). In

Tzouros and Pähler

Figure 2. SDS-PAGE of proteins from RLMs incubated with a 1:1 mixture of 1/14C1-1 and bands 1-3 selected for identification. (A) Coomassie staining and (B) autoradiography, 6 days of exposure.

fact, no LC-MS signal for the identified target peptides was detected anymore if samples were left in a solution of 5% formic acid for 2-3 days (data not shown). Enrichment of Modified Peptides by SCX Chromatography. A further analytical challenge, the selective enrichment of modified peptides out of the bulk of unmodified ones, was overcome by SCX chromatography that separates peptides according to their solution net charge. SCX is a widely used prefractionation step in multidimensional analyses of complex protein digests (36, 37). Typically, trypsin produces peptides with two basic sites, the nitrogen-containing side chains of a C-terminal lysine or arginine and the N-terminal amino group (Figure 3A). Thus, under low pH conditions of SCX chromatography such as pH 3, these two sites are protonated, and the solution net charge is mainly 2+. Furthermore, in case of a protein modification with a nitrogen-containing substrate found in many druglike lipophilic bases (e.g., compound 1), covalent modification of an amino acid results in the availability of additional sites for protonation. Cysteines residues are known for efficient trapping of reactive metabolites due to the nucleophilic nature of the thiol group (34). As a consequence, the solution net charge of the modified peptide is expected to increase from 2+ to 3+. Because of the higher charge, the affinity of the peptide for the SCX resin is increased, and it is expected to elute in later fractions as compared to the bulk of the 2+ unmodified tryptic peptides. This approach was further supported by an in silico tryptic digestion of the entire rat proteome, as predicted through mapping of known rat, mouse, and human proteins to the rat genome draft. The theoretical charge state of the individual in silico predicted peptides was calculated as the sum of their protonable residues, that is, the N terminus, the C-terminal lysine or arginine, added to any histidine. As a result, 72% of potential tryptic peptides were predicted to have a net charge of 2+ (Figure 3B). Furthermore, regarding the subset of peptides containing cysteine residues, 68% are predicted to be doubly charged if one cysteine is present. Desalted peptides were fractionated by two successive offline SCX runs, where 80% of the sample was used for determining radioactivity and the remaining 20% for the nanoLCMS/MS experiments (see the Experimental Procedures and Scheme 1). The SCX chromatogram of the protein digest of band 3 revealed



Figure 3. SCX enrichment strategy of modified peptides. (A) Additional sites available for protonation after Cvb of a reactive metabolite of drug candidate 1. Tryptic peptides at pH 3 increase their net charge to 3+. The experimental pKa values for 1 indicate that the pyridinyl group is the most likely acceptor. x denotes the position of any amino acid. (B) Theoretical net charge distribution obtained for an in silico tryptic digestion of the translated rat genome. A total of 595168 peptides (MW 400-3000) were predicted.

Figure 4. SCX chromatogram of the in-gel tryptic digest of band 3 monitored by UV at 210 nm and radioactivity. The charge state distribution is shown for the peptides identified by a database search (see Figure S-1 in the Supporting Information). The majority of the unmodified peptides elutes as 2+ charged species in the early fractions. Arrows indicate fractions searched for modified peptides.

three fractions, namely, 8, 15, and 21, that contained significant radioactivity related to the presence of modified peptides (Figure 4). The charge state distribution obtained by the SCX separation was determined by analyzing the different fractions by nanoLCMS/MS and peptides identified by searching the data against a protein database. Doubly charged peptides eluted in fractions 5-24, while triply and quadruply charged species were found in fractions 12-28 and 19-30, respectively (data provided in Figure S-1 in the Supporting Information). The highest level of radioactivity observed was attributed to fraction 15, effectively located in a region of the chromatogram where triply charged peptides were observed. In fact, the charge state of the majority of the modified peptides is 3+, since the presence of the modification serves as an additional site for protonation. Furthermore, selective enrichment of modified peptides was accomplished since the majority of the tryptic peptides eluted in earlier fractions. Indeed, intense UV signals are observed around fractions 5 and 8, corresponding to 2+ species. Identification of Modified Peptides and Protein Targets. The radioactivity containing SCX fractions were analyzed by nanoLC-MS/MS to detect and identify modified peptides (see Scheme 1). Because the incubation was performed with a 1:1 mixture of 1/14C1-1, all modified peptides are detected as an isotopic doublet that differs in mass by 2 Da. More specifically, because peptides are typically observed as multiply charged

species when ionized by electrospray, the isotopic doublet is expected to have a ∆m/z of 1.002 and 0.668 for doubly and triply charged ions, respectively (see simulated isotopic pattern in Figure 5B). Searching the LC-MS chromatogram of SCX fraction 15 from protein band 3 for candidates fitting the isotopic doublet criteria, we were able to extract the triply charged ion with m/z 571.2433 eluting at tR 29.2 min (Figure 5). It should be mentioned that fraction 15 contained a second triply charged ion with m/z 565.9117 later identified as the same peptide but with a different metabolite containing one oxygen atom less (-16 Da). The MS and MS/MS data for this second modified peptide are provided (Figures S-2 and S-3 in the Supporting Information). The modified peptide with m/z 571.2433 was identified to contain the sequence VFANPEDCAGFGK on the basis of the MS/MS data, with the reactive metabolite 1 + 3O + 4H attached to the cysteine residue and corresponding to the elemental formula C76H102N20O24S (see the MS/MS interpretation below). For verification, the theoretical [M + 3H]3+ isotopic pattern of the peptide together with its 14C-labeled analogue possessing the elemental formula 14C1C75H102N20O24S, and present in a 1:1 ratio, was plotted (Figure 5B). Beside the good 1.89 ppm mass accuracy for the measured ions, the relative intensities of the various isotope peaks were in good agreement. It should be mentioned that the intensities of the two parent peptide ions

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Figure 5. nanoLC-MS of SCX fraction 15 from gel band 3. (A) Base peak and extracted ion chromatograms of the triply charged ion m/z 571.24. (B) Measured (top) and simulated (bottom) full scan MS of the peptide ion m/z 571.2433 with a reactive metabolite attached as a 1:1 mixture of 12 C and 14C. The simulated spectrum was generated after identification of the peptide corresponding to the elemental formula C76H102N20O24S.

are not identical, since the natural heavy isotopes of the unlabeled species add to the one observed for the 14C1-labeled ions. This unique isotope fingerprint can be used as an MS signature to enable chromatogram searches in an automated way, employing computer-assisted isotope pattern recognition software. For that purpose, the lowest number of false-positive hits was obtained when five consecutive isotopic peaks were considered for the isotopic cluster and used as search parameters. In the current version of the software used, no ion charge state could be defined, so that successive searches had to be performed when looking for doubly and triply charged species. The characteristic 12C/14C isotopic pattern is not limited to straightforward recognition of parent ions in the MS, since it can also be observed at the MS/MS level. Given that the covalent bond between the amino acid and the modification is stable under CID conditions, metabolite carrying fragment ions will display the same characteristic isotopic signature in the MS/ MS. Recognition and structure assignment of modified fragment ions are further facilitated when detected with high resolving power and accuracy in the Orbitrap. For instance, the MS2 experiment of the modified peptide at m/z 571.2433 produced four such fragment ions, detected at m/z 342.1202, 392.1025, 733.2936, and 1123.4657 (Figure 6A,B). The singly charged fragment ion at m/z 392.1025 turned out to be of particular interest: It corresponded to the metabolite part with the sulfur atom of the cysteine residue attached. On the basis of the m/z value and a mass shift of +52 Da, the attached metabolite could be characterized as the result of a formal gain of three oxygen atoms and four protons, consequence of multiple oxidation and hydrolysis steps. The ion at m/z 392.1025 can thus be described as [1 + 3O + 5H + S]+ with an excellent mass accuracy of 0.21 ppm. Unfortunately, attempts to obtain a more detailed picture of the metabolite, for example, by submitting m/z 392.10 to MS3, were unsuccessful. Another example is reflected by the fragment ion recorded at m/z 733.2936: The MS analysis in the Orbitrap clearly revealed that apart from containing the metabolite in its structure, it was doubly charged and could be firmly assigned to y112+ with 0.41 ppm accuracy (see Figure 6B). Apart from the previously highlighted fragment ions, very few peptide sequence information could be gathered from the MS2 experiments. Nevertheless, the intense doubly charged species detected at m/z 660.8148, complementary to the ion at m/z 392.1025, did correspond to the peptide part and was selected for MS3 experiments (see Figure 6B). Indeed, the shape

of the ion isotopic distribution further supported this assumption. Because CID resulted in abstraction of the sulfur atom from the Cys, the residue had now to be considered as a dehydroalanine (dAla) for the peptide moiety (Figure 7). Such a fragmentation route, where sulfur is transferred from the peptide to the adducted entity, is frequently observed for GSH reactive metabolite conjugates (9, 38). The MS3 of m/z 660.81 resulted in extensive peptide backbone cleavage, clearly indicating the position of the dAla residue. Indeed, peptide consecutive sequence ions y6 (m/z 548) and y5 (m/z 479), resulting in a ∆m/z of 69 amu left no doubt about the site of modification. Interpretation of the MS/MS data led to the identification of the sequence VFANPEDCAGFGK, corresponding to residues 43-55 and unique peptide of rat microsomal glutathione S-transferase 1 (MGST1). Furthermore, Cys50 was unambiguously demonstrated as the residue modified by reactive metabolites of 1. The peptide VFANPEDCAGFGK was detected in two other variations, that is, with two other metabolites covalently linked to Cys50, explaining the high intensity of band 3 in the autoradiography (see Figure 2B). The complete nanoLC-MS/ MS data of these two peptides are provided as Supporting Information (Figures S-2-S-5 in the Supporting Information). The same methodology was applied for the characterization of modified proteins present in gel bands 1 and 2 (see Figure 2). It resulted in the identification of three peptides that were unique to two proteins, dimethylaniline monooxygenase (N-oxideforming) 1 (FMO1) and P450 2C11 (a male rat specific enzyme), respectively. Complete nanoLC-MS/MS data are provided as supplemental figures (Figures S-6-S-8 and S-9-S-10 in the Supporting Information for FMO1 and P450 2C11, respectively). The list of all identified modified peptides is presented in Table 1.

Discussion We have developed and optimized a method to unambiguously identify peptides and their parent proteins modified by reactive drug metabolites. It allows us to precisely map the modified proteins, including characterization of the modified residue and nature of the bound reactive metabolite. The key element of the strategy is represented by the use of an equimolar mixture of non- and radiolabeled substrate for the in vitro experiments. More than just a way to track modified proteins and peptides throughout the multidimensional separations from complex samples, the substrate mixture provides a unique MS



Figure 6. CID fragment ion spectra of m/z 571.24. (A) MS2 of m/z 571.24 acquired in the Orbitrap. Asterisks denote fragments exhibiting the 12 14 C/ C isotopic pattern. (B) Magnified view of fragment ions m/z 392.1025, 660.8148, and 733.2936. (C) MS3 of m/z 571.24/660.81 acquired in the LTQ. The modified residue indicated by the 69 amu difference between y6 and y5 corresponds to a dAla.

Figure 7. Proposed main CID fragmentation route of MGST1 peptide VFANPEDCAGFGK covalently modified by the reactive metabolite 1 + 3O + 4H on the cysteine residue.

isotope fingerprint for the unambiguous identification and characterization of modified peptides. The proof of concept was established using a model compound known to induce high Cvb to human and rat hepatic

proteins and also to time dependently inactivate human P450 3A4. Using RLMs as an enzyme source, three proteins, namely, MGST1, FMO1, and P450 2C11, were highlighted and demonstrated to be modified by reactive metabolites of compound 1. Performing incubations with a mixture of non- and radiolabeled substrate for Cvb studies allowed pinpointing targets of reactive metabolites down to the modified amino acid. These detailed findings were achieved using a proteomics workflow combining autoradiography of SDS-PAGE separated proteins, in-gel digestion, and SCX enrichment followed by nanoLC-MS/MS analysis of the resulting peptides. It has to be pointed out that, beside the possibility to trace radioactive protein bands, the underlying “GeLC-MS/MS” approach is well-suited to analyze microsomal proteins. In fact, microsomes are known to contain hydrophobic membrane-bound proteins such as cytochrome P450 and other drug-metabolizing enzymes that are best solubilized by using SDS as a detergent (35, 39, 40). Another key step in the analysis workflow was the peptide fractionation step by SCX chromatography. More than only adding a second chromatographic dimension, SCX proved to operate as an enrichment tool for modified peptides. Provided the reactive metabolites act as a base when adducted to a peptide, these species can accommodate an additional charge and be

860


Tzouros and Pähler

Table 1. Peptides Covalently Modified by Reactive Metabolites of Compound 1 Identified from Rat Liver Microsomal Proteinsa proteinb band

SCX

1

24 24 25 8 15 15

2 3

name

peptide c

monoisotopic elemental mass d

accession no.

MW

sequence

Cys

bound metabolite

measured

calcd

error (ppm)

FMO1

P36365

59825

P450 2C11 MGST1

P08683 P08011

57181 17472

R.SCDLGGLWR.F R.CalkIYFNTKVCSITK.R R.SPCMKDR.S K.VFANPEDCAGFGK.G K.VFANPEDCAGFGK.G K.VFANPEDCAGFGK.G

35 111 338 50 50 50

1 + 2O + 4H 1 1 - O - 2H 1-alkyl + 2O + 4H 1 + 2O + 4H 1 + 3O + 4H

1346.5802 1880.8676 1122.4469 1589.6560 1694.7119 1710.7064

1346.5821 1880.8702 1122.4482 1589.6569 1694.7147 1710.7097

1.58 1.36 1.14 0.59 1.66 1.89

a The bold entry is discussed in detail in the text. b From the Swiss-Prot/TrEMBL database (http://www.expasy.org/sprot/); P450 2C11 is recorded as CP2CB_RAT. c The modified Cys is underlined; Calk ) carbamidomethylated Cys. d Proposed transformation.

isolated from unmodified tryptic peptides due to a higher affinity for the SCX resin. It must be stated that this particular property did not apply to all observed peptide adducts. An example was also found where Cvb and peptide modification by a metabolite of 1/14C1-1 did not lead to a general increase of the solution charge state. For instance, the peptides found in fraction 8 (see Figure 4) were determined to bear a metabolite that had lost the pyridinyl-dimethyloxy group via metabolic dealkylation. Such a reaction was also observed for the main GSH adduct of 1, since that same side chain of the metabolite was cleaved (see proposed structure in Figure 1). As a consequence, no additional protonation and charge shift from 2+ to 3+ was observed. This species appeared to have low affinity for any additional H+, further supporting that the 2-amino-pyrimidine group does not act as a base in the present conditions (see Figure 3A). Another set of modified peptides, eluting in fraction 21, located in the 4+ region, was examined. However, they were found to share the same core sequence as the ones detected in fraction 15 but with an additional five amino acids at the C terminus due to a trypsin missed cleavage (VFANPEDCAGFGKGENAK vs VFANPEDCAGFGK). Because they contained an additional basic residue in the peptide backbone, namely, a second lysine residue, these peptides were present as quadruply charged species at pH 3 and eluted in the very late fractions. Apart from being a radioactivity-tracking tool, the occurrence of 12C/14C-modified peptides within a mixture of unchanged ones was precious as they could be differentiated by MS. In particular, modified peptides were found to exhibit a characteristic isotope pattern due to attached 12C/14C metabolites. The detection step could be automated using isotope pattern recognition software, while peptide sequence and metabolite structure had to be determined manually. It must be emphasized that direct use of peptide MS/MS identification software, using a predicted mass for the bound metabolite of 1, failed in the present investigation. Although it was reported that this traditional way of mining data successfully allowed one to obtain positive search hits for certain studies (23, 25), it did not suggest any reliable modified peptide identification in our case. This may be due to the fact that the attached metabolite is readily cleaved from the peptide under CID conditions, generating complex MS/MS spectra with more fragment ions than those expected from peptide backbone fragmentation alone. Moreover, the later peptide backbone ions can be shifted, depending on the bond cleavage between the metabolite and the modified residue. For instance, we observed the modified cysteine of VFANPEDCAGFGK to be transformed to a dAla when attached to a particular metabolite of 1 (see Figure 6). There is no way such an MS/MS would result in a reliable identification using a peptide sequencing software. In other cases where metabolites have a labile structure, the MS/ MS can be dominated by strong ions originating from the metabolite itself and suppress the more informative ones expected for the peptide. For instance, neutral losses of water or ammonia deriving from the metabolite are sometimes the

preferred fragmentation pathways of modified peptides (see Figures S-3, S-7, and S-8 in the Supporting Information). Despite the fact that reactive metabolites are susceptible to be linked to any electrophilic residue (Cys, Lys, His), the identification strategy also has the advantage of being unrestrictive in the amino acid that must be considered as a modification site. In contrast, when using MS/MS data-mining softwares, suggestions about potential modification sites are requested to result in any peptide identification. Application of our strategy to the drug candidate 1 in RLMs unambiguously revealed three proteins involved in xenobiotic metabolism, the transferase MGST1 and the two oxidoreductases FMO1 and P450 2C11. Unfortunately, repeating the same workflow for other minor radioactive bands located in the 55-98 and 22-36 kDa region (see Figure 2) did not succeed in the identification of more protein targets. Despite clear Cvb at the protein level, very low radioactivity was recorded for the SCX fractions of the tryptic digests. Thus, no additional efforts were made for these bands by LC-MS. To validate our method and the three targets, the presence of additional peptides originating from MGST1, FMO1, and P450 2C11 was questioned. LCMS/MS data obtained from the entire tryptic digests of proteins contained in bands 1-3 (see Figure 2) were searched against a protein database. As a result, MGST1 was confirmed to be one of the major proteins present in band 3, since 13 peptides could be identified corresponding to an amino acid sequence coverage of 68% (Table S-1 in the Supporting Information). The same applied to FMO1 and P450 2C11, resulting in 20 and 32 peptides identified and sequence coverages of 41 and 50%, respectively (FMO1, Table S-2, and P450 2C11, Table S-3 in the Supporting Information). Having this information in hand, we searched the Target Protein DataBase (TPDB, release of April 17, 2008, 268 entries) (41) for earlier reports on Cvb involving the three identified proteins. As a single result, P450 2C11 was cited to be a target of diclofenac in a study involving in vivo/vitro experiments (42). In this case, detection was performed by immunostaining methods; thus, no information about metabolite(s) structure and site(s) of modification could be specified. Concerning MGST1 and FMO1, no chemical was reported to form adducts with these proteins in rats. Using a different species and model biotinylated reactive electrophiles, the homologous human MGST1 was identified to be a target in HLMs (25). A detailed investigation by MS showed that Cys50 was the modified residue for the human MGST1, identical to the one highlighted in the present work for the rat protein (see Table 1). Functional studies on MGST1 have already shown the importance of Cys50 since it serves as a switch whose alkylation modulates enzyme activity (43, 44). The accurate mass measured for the peptide adducts further allowed deducing the type of reactive metabolites attached to the microsomal proteins. Five different reactive metabolites of 1 were identified probably resulting from initial oxidation of the furan ring (see Table 1 and Figure 1). The presence of modified peptides exclusively


at the cysteine residues argues in favor of the furan epoxide rather than the γ-ketoenal activation step. Also, no adducts to amino groups such as lysine residues were observed. Some of the bound metabolites appeared to be highly oxidized species, for example, an FMO1 and an MGST1 peptide with 1 + 2O + 4H, probably containing a second oxidation spot remote from the furan ring such as in the pyridinyl group or the aliphatic carbons. Such multiple P450-catalyzed biotransformations have already been described for other furan-containing compounds (45). Beside the MS-based evidence for the presence of covalent peptide adducts, the detailed metabolism of 1 was not further explored in this context and will be the subject of a separate study. This methodology has the potential to be applied to study other substances inducing protein Cvb. Furthermore, the presented analytical route using liver microsomes as the investigated biological matrix can be easily applied to other types of samples such as other in vitro systems like primary hepatocytes or to tissues samples from in vivo studies. Employing such more physiological systems would allow for an even more complete overview of the extent and specificity of protein Cvb in the case of drug bioactivation. Although liver microsomes are a well-characterized system allowing for the characterization of bioactivation potentials (8), covalent protein binding in human hepatic microsomes only allows for identification of a potential hazard but does not characterize the risk for elucidation of toxicity. There is compelling evidence in the literature that excessive protein Cvb in combination with high clinical dose is certainly a high risk associated with cases of idiosyncratic toxicity (2, 46-49). Still, none of the preclinical assays to characterize reactive metabolite formation can be regarded as prospective tools to predict potential toxicities in the clinical setting. Obach et al. have recently demonstrated that the utilization of in vitro Cvb data in HLMs to prospectively predict toxicity is not successful to predict toxicity outcomes (50). Even the incorporation of the different dose levels and extents of metabolism were not particularly helpful to distinguish hepatotoxic from nonhepatotoxic compounds. It is our current belief that Cvb assessment in primary hepatocytes reflects a better balance between competing metabolic pathways that lead to activation; scavenging of reactive metabolites but also competing phase II activation and mostly detoxification pathways are better suited to characterize a Cvb hazard than assays in hepatic microsomes. A comparative study to assess the potential to distinguish hepatotoxic and nonhepatotoxic compounds based on the human daily dose, fraction absorbed, and Cvb properties in primary human hepatocytes is currently ongoing in our group. Additionally, there is little known about the sequence of events between protein adduct formation and toxicity. On a mechanistic point of view, following up the present findings, it would be of great interest to further investigate protein targets of reactive metabolites of compounds shown to induce clinical signs of toxicity to establish a link between protein targets and toxicity. Acknowledgment. We thank Dr. Martin Ebeling from the Group Research Information at F. Hoffmann-La Roche Ltd. for generating the in silico rat proteome digest and the peptide predictions and Drs. Bjo¨rn Wagner and Holger Fischer from the Molecular Properties group Discovery Chemistry for the pKa determination. We also thank Drs. Ellen Pa¨hler and Christoph Funk for critical review of the manuscript. Supporting Information Available: Figure containing peptide charge state distribution during SCX and tables containing


identified peptides for MGST1, FMO1, and P450 2C11 using SEQUEST as well as additional modified peptide MS and MS/ MS data as mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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TX800426X

A Targeted Proteomics Approach to the Identification of Peptides

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