Sequence Mapping of Epoxide Adducts in Human Hemoglobin with

use of LC-MS-MS and the SALSA algorithm as a general approach to map xenobiotic adducts on proteins at the level of amino acid sequence. Hemoglobin (H...
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Chem. Res. Toxicol. 2002, 15, 799-805

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Sequence Mapping of Epoxide Adducts in Human Hemoglobin with LC-Tandem MS and the Salsa Algorithm Hamid Badghisi and Daniel C. Liebler* Department of Pharmacology and Toxicology, and Southwest Environmental Health Sciences Center, and College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207 Received November 27, 2001

The rapid development and integration of liquid chromatography-tandem mass spectrometry (LC-MS-MS) has enabled the high-throughput identification of proteins and driven the expanding field of proteomics. LC-MS-MS also offers an attractive general approach to the analysis of xenobiotic adducts on proteins. The aim of this study was to examine the combined use of LC-MS-MS and the SALSA algorithm as a general approach to map xenobiotic adducts on proteins at the level of amino acid sequence. Hemoglobin (Hb) adducts are commonly used as biomarkers for exposure to environmental toxicants. Human Hb was incubated with styrene oxide, ethylene oxide, and butadiene dioxide (40 mM) to form adducts, digested with trypsin and analyzed by LC-MS-MS on a ThermoFinnigan LCQ ion trap MS instrument. Datadependent scanning was used for acquisition of MS-MS spectra. The SALSA algorithm was used to detect MS-MS spectra of native and modified Hb peptides. The adducted sites identified are the N-terminal valines of both HbR and Hbβ, glutamic acid 7, cysteine 93, and histidines 77, 97, and 143 of the β chain and histidine 45 of the R chain. Specific shifts in the b- and y-ion series in MS-MS spectra confirmed the locations of each adduct. This approach offers a means to simultaneously identify multiple Hb adducts resulting from exposures to known or unknown toxicants. Combined application of LC-MS-MS and SALSA thus provides a general means of mapping protein modifications at the level of amino acid sequence. The rapid advancement and integration of MS,1 protein and peptide separations, and data mining tools has been instrumental in the emergence of proteomics (1, 2). Proteomics approaches offer new opportunities to understand the mechanisms of chemical toxicity and new means of assessing chemical exposures. An important problem in toxicology is the characterization of protein targets of reactive electrophiles and the impact of covalent modifications on protein functions (3, 4). Tandem MS-based analyses have been applied previously to identify adducts on xenobiotic-modified proteins (5-11). Although these studies clearly established that tandem MS data can provide unequivocal sequencespecific location of adducts, successful analyses required prior knowledge of the chemical nature and masses of the adducts, as well as painstaking manual analyses of the MS-MS spectra. The problem of mapping adducts on proteins becomes considerably more complicated when multiple protein targets are considered or when multiple known or unknown species may modify proteins. Recent work in our laboratory has focused on the development of generally applicable methods to identify protein targets of reactive intermediates and to map the adducts at the level of amino acid sequence. Our approach involves automated LC-MS-MS analysis of pro* To whom correspondence should be addressed. Telephone: (520) 626-4488. Fax: (520) 626-6944. E-mail: [email protected]. 1 Abbreviations: BDE, butadiene diepoxide; EO, ethylene oxide; CID, collision-induced dissociation; Hb, hemoglobin; LC-MS-MS, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; MS-MS, tandem mass spectrometry; SO, styrene oxide; TFA, trifluoroacetic acid.

teolytic digests from mixtures of adducted and unadducted proteins with data-dependent instrument control to obtain MS-MS spectra of peptides without regard to modification status. Subsequent analysis of the MS-MS data with the Sequest program (12) identifies proteins represented by MS-MS spectra in the datafiles. Further analysis with the SALSA algorithm identifies MS-MS spectra that may display adduct-specific fragmentation characteristics (13). We have recently shown that the SALSA algorithm can be used to mine datafiles to identify MS-MS spectra from specific peptide sequence motifs (14). This application of SALSA can detect spectra of modified and unmodified peptides based on similarities between ion series generated by fragmentation of peptides that share sequence homology. Hemoglobin adducts formed by epoxides of styrene, ethylene, and butadiene are among the most widely studied markers of exposure to these chemicals (for reviews, see refs 15-17). Indirect methods of analysis suggested that adducts with cysteine and glutamate are formed with aliphatic epoxides (18-20). However, only the N-terminal valine adducts have been unambiguously measured by a modified Edman degradation procedure (21, 22). Application of this method to specific adducts requires synthesis of stable isotope-labeled standards for each type of adduct to be investigated. Kaur and colleagues employed fast atom bombardment tandem MS to identify a histidine adduct of styrene oxide in human Hb (23). More recently, Moll et al. (24) employed electrospray MS to identify peptides adducted with butadiene monoxide in mouse erythrocytes in vitro. This was the

10.1021/tx015589+ CCC: $22.00 © 2002 American Chemical Society Published on Web 05/10/2002

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first systematic application of MS to comprehensive mapping of Hb adducts. However, the analyses did not include tandem MS to provide sequence-specific mapping of adducts. Here we describe the application of LC-MS-MS and the SALSA algorithm to the sequence-specific mapping of aliphatic epoxide adducts formed with human hemoglobin in vitro. In our previous work, we found that sequence motif searches not only identified MS-MS spectra of the target peptides but also spectra of peptides containing oxidized amino acids. However, we had not previously extended this approach to mapping larger xenobiotic adducts on proteins. MS-MS spectra provide fragmentation fingerprints for both unmodified and epoxide-adducted Hb peptides. The application of the SALSA algorithm identifies MS-MS spectra that contain ion series consistent with Hb peptides or their variant forms. Subsequent inspection of the MS-MS scans identified with SALSA confirms the target peptide sequence and reveals the position of adduct modification. The data confirmed previously identified hemoglobin-epoxide adducts and identified several others that had not been reported previously. The results indicate that LC-MSMS and SALSA provide a generally applicable approach to sequence specific mapping of xenobiotic adducts on proteins.

Materials and Methods Caution: Ethylene oxide, styrene oxide, and butadiene diepoxide are highly toxic; special care must be taken while handling these chemicals. Chemicals. Styrene oxide, 1-propanol, and trifluoroacetic acid (TFA) were obtained from Aldrich (Milwaukee, WI). Ethylene oxide was obtained from Supelco (Bellefonte, PA). Butadiene diepoxide, hemoglobin, urea, and ammonium bicarbonate were purchased from Sigma (St. Louis, MO). All other chemicals were of HPLC grade, reagent grade or higher purity. Hb Adduct Formation. Hemoglobin adducts were prepared by adaptation of the procedure of Pauwels et al. (22). To 0.5 g of human hemoglobin dissolved in 3.5 mL of isotonic saline were added styrene oxide, ethylene oxide, and butadiene diepoxide to final concentrations of (40 µM to 40 mM) from stock solutions in methanol. Following 6 h of incubation in a shaker at 37 °C, the reaction was stopped with 1-propanol containing 0.05 M HCl. After centrifugation at 3000g, the globin was precipitated with ethyl acetate and the excess epoxides were removed by washing the samples twice with 10 mL of ethyl acetate and twice with 10 mL of benzene. The protein residues were then dried on filter paper and stored at -20 °C until further analysis. Digestion of Globins. The dried globin samples were digested with porcine sequencing grade modified trypsin (Promega, Madison, WI) at an enzyme-to-substrate ratio of 1:25 (w/w). The dried globin was dissolved in 8 M urea, 0.4 M NH4HCO3, and 45 mM dithiothreitol, and incubated in a 37 °C water bath for 24 h. The digestion was stopped by quickly freezing the sample and storing at -20 °C. Mass Spectrometry and Data Acquisition. The globin digest was diluted with acetonitrile/water (1:1, v/v) and analyzed by LC-MS-MS on a Thermo Finnigan LCQ ion trap mass spectrometer equipped with a ThermoFinnigan electrospray ionization source. Chromatography was carried out on a Vydac protein and peptide C-18 column (250 × 1 mm) at a flow rate of 15 µL min-1 with a gradient of 3 to 95% acetonitrile: (0.5% formic acid /0.01% TFA). Data-dependent scanning was used to achieve automated acquisition of MS-MS spectra of peptide ions. In the data-dependent scan experiments, the instrument was set to conduct repeated cycles of one MS-MS scan followed by three successive MS-MS scans of selected precursor ions. The dynamic exclusion mass was set to (1.5 m/z, with ions residing

Badghisi and Liebler

Figure 1. Generic structures for peptide adducts derived from styrene oxide, ethylene oxide and butadiene dioxide. The modified sequence shown is the N-terminal tryptic peptide of Hbβ. on the exclusion list for 5 min. Data analysis of the tandem MS experiment was performed with ThermoFinnigin Xcalibur version 1.2 software. SALSA analyses of MS-MS data were done with a prototype SALSA program written in Visual Basic and running under Windows 2000 on a 500 MHz Pentium PC. MS-MS scans corresponding to specific Hb peptide sequences or their modified variants were identified by sequence motif analysis of LC-MSMS datafiles with the SALSA algorithm (14) (see Results, below).

Results Overview and Rationale for Approach. The objective of these studies was to apply LC-MS-MS and the SALSA algorithm to map epoxide adducts in human hemoglobin at the level of amino acid sequence. Hb was chosen as a model protein for this work because of the easy availability of the purified protein and because previous work had shown that Hb is a target for adduction by aliphatic epoxides (for reviews, see refs 15-17). The three epoxides used as model alkylating agents for these studies have been shown previously to react with hemoglobin in vitro and in vivo. Adducts formed in vivo have been used as biomarkers of exposure to the epoxides or their parent compounds. Generic structures of the three peptide adducts are depicted in Figure 1. The analytical approach taken in these studies was to generate adducts by incubation of Hb with the epoxides in vitro, to digest the adducted proteins, and to analyze the peptide digests by LC-MS-MS with data-dependent scanning, which generates datafiles containing MS-MS spectra of both modified and unmodified peptides. Data analysis with the SALSA algorithm allows identification of MS-MS spectra that correspond to specific peptide sequences or their modified forms (13, 14). Fragmentation of a peptide ion in MS-MS gives rise to series of band y-ions whose spacing along the m/z axis of the spectrum indicates peptide sequence. Both modified and unmodified forms of the peptides fragment to yield b- and y-series ions and adduct modifications may shift some of these ions to different absolute m/z values. However, the relative relationships of some of these ions along the m/z axis are preserved in MS-MS spectra of the modified peptides, thus enabling their detection by SALSA. A SALSA analysis of the MS-MS data thus identifies those

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Figure 2. MS-MS spectrum for the doubly charged styrene oxide adduct of the N-terminal tryptic peptide of Hbβ (m/z 537.4). The adduct is on the peptide N-terminus as indicated by b-ions that appear at 120 amu greater than in the unmodified peptide.

MS-MS scans most likely to correspond either to the unmodified peptide or adducts of that peptide. To map adducts throughout the HbR and Hbβ chains, a list of tryptic peptides was generated and then SALSA searches were done with search motif sequences corresponding to the central portion of each peptide. MS-MS spectra receiving the highest scores in each datafile then were inspected manually to verify correspondence between the spectrum and the target peptide sequence, as well as the locations and masses of modifications. Analysis of Hemoglobin-Styrene Oxide Adducts. Human hemoglobin was incubated with 40 mM styrene oxide in isotonic saline for 6 h at 37 °C and the hemoglobin then was recovered, digested with trypsin, and the peptides were analyzed by LC-MS-MS. Analysis of the datafile with Sequest indicated that MS-MS spectra assigned to expected tryptic peptides yielded 79% coverage (by amino acid sequence) for HbR and 86% coverage for Hbβ (not shown). Systematic SALSA searches of tryptic peptide sequence motifs identified MS-MS spectra of both unmodified and modified peptides. The results of a SALSA search for MSMS spectra corresponding to the N-terminal tryptic peptide VHLTPEEK (residues 1-8) of Hbβ are shown in Figure 2. The search motif HLTPEE was used to search for MS-MS spectra containing ion series that matched the expected y-ion series for the peptide (14). SALSA awarded scores of >20 to 17 MS-MS spectra, all of which contained ion series matching the search motif. The highest scoring MS-MS scans (scans 2110-2112, SALSA score 49.79) resulted from CID of a precursor ion of m/z 537.37, which corresponded to the doubly charged ion of the styrene oxide monoadduct (calculated avarage m/z 537.14). Inspection of these MS-MS spectra indicated the expected y-ion series for the target peptide, whereas the b-series ions were shifted by +120 amu, which corresponds to the mass of the styrene oxide adduct (Figure 2). This information unambiguously established the location of the adduct at the peptide N-terminus. Additionally, the presence of a peak at m/z 853.3 corresponds to unmodified y7-ion, thus providing additional support that the modification is at the N-terminal valine, rather than on the adjacent histidine residue. The doubly charged peptide adduct also was detected in MS-MS scans 1967-1969, 2150-2152, 2002-2004, and 1951-1953 with SALSA scores of 43.98, 41.49, 34.98, and 28.48, respectively. SALSA also detected MS-MS scans for the singly charged ion at m/z 1074.6 (calculated

average m/z 1073.28) of the styrene oxide adduct (scans 1959-1961, SALSA score 36.36). Note also that the MSMS spectrum of the doubly charged ion of the styrene oxide adduct displays a prominent neutral loss of 120 amu, which corresponds to the styrene oxide moiety. Adduct-specific neutral and charged losses as well as adduct-derived product ions are characteristic features of MS-MS fragmentation of peptide adduct ions (13, 14, 25-27). The unmodified Hbβ 1-8 peptide was detected by MSMS of the singly charged ion (calculated average m/z 953.08) in MS-MS scans 1466-1468, 1494-1496, 15071509, and 1426-1428 with SALSA scores of 47.73, 40.68, 40.53, and 38.39, respectively. The doubly charged, unmodified peptide (calculated average m/z 477.05) was detected in MS-MS scans 1482-1484, 1450-1452, and 1490-1492 with SALSA scores of 33.40, 33.30, and 31.07, respectively. Further inspection of the SALSA output also indicated two unanticipated variants of the target peptide. The first was from CID of m/z 491.27 (SALSA score 39.43) and corresponded to a modification of +28 amu, probably due to an N-terminal formylation. The second was from CID of m/z 499.47 (SALSA score 38.54) and corresponded to a modification of +43 amu, probably due to an N-terminal carbamylation. Inspection of the MS-MS spectra revealed the expected shifts in the b-series ions and verified the assignments as N-terminal modifications (data not shown). The formylation may have been in the original protein sample or may be due to formic acid in the mobile phase, whereas the N-terminal carbamylation is frequently observed in analyses of peptides from enzymatic digests done in the presence of urea (28, 29). Replacing urea with guanidine eliminated these carbamylations. Analysis of Hemoglobin Adducted with Mixtures of Styrene Oxide, Ethylene Oxide, and Butadiene Dioxide. The analysis of data from the above experiment illustrates the general approach employed in using the SALSA algorithm to detect peptide adducts from LC-MSMS data. We next extended this approach to map hemoglobin adducts formed by a mixture of aliphatic epoxides. Hemoglobin was incubated with a mixture of styrene oxide, ethylene oxide and butadiene dioxide (all at 40 mM) in isotonic saline at 37 °C for 6 h. The protein mixture then was digested with trypsin and analyzed by LC-MS-MS with data-dependent scanning. A systematic search of hemoglobin peptides for adducts entails SALSA searches for sequence motifs in all pos-

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Table 1. Summary of SALSA Results of MS-MS Scans from Analysis of Tryptic Peptides of Hb Adducted by Styrene Oxide, Ethylene Oxide, and Butadiene Dioxide Hb chaina

target peptideb

β(1-8)

VHLTPEEK

β(67-82)

VLGAFSDGLAHLDNLK

β(83-95)

GTFATLSELHCDK

β(96-104)

LHVDPENFR

β(133-144)

VVAGVANALAHK

R(1-7)

VLSPADK

R(17-31)

VGAHAGEYGAEALER

SALSA score

m/zc

identity

38.24 40.63 33.15 25.98 4.21 36.62 16.54 11.66 13.81 7.44 6.48 4.41 19.93 12.95 14.8 11.13 26.04 49.12 39.56 32.76 25.55 14.53

476.96 537.24 520.11 498.39 529.15 835.83 896.41 712.11 771.72 763.8 754.57 771.79 564.17 624.23 575.73 635.78 729.46 425.49 774.46 766.25 826.86 787.29

unmodified +SOd +BDE +EO +BDE (104) unmodified +SO unmodified +SO +BDE (104) +BDE unknowna unmodified +SO unmodified +SO unmodified +SO +EO unmodified +SO +EO

a Hb chain and amino acid positions in the target peptide. b The peptide sequence used as a SALSA search motif is underlined. The adducted amino acid is indicated in boldface type. c The m/z of the precursor ion is listed. d The modifications are SO, styrene oxide (M + 120); EO, ethylene oxide (M + 44); BDO1, butadiene dioxide adduct 1 or 3 (Figure 1) (M + 86); BDO2, butadiene dioxide adduct 2 (Figure 1) (M + 104).

sible tryptic peptides. The success of such an approach is highly dependent on the acquisition of MS-MS spectra for as many of the tryptic peptides as possible. Analysis of the LC-MS-MS datafile for the digest of the alkylated hemoglobin with Sequest indicated that 79% of the HbR and 80% of the Hbβ sequences were assigned to MS-MS scans. The Sequest Xcorr scores for the MS-MS spectra assigned to specific sequences all exceeded 2.50 and inspection of the spectra verified correspondence between predicted b- and y-ions and the major peaks in the spectra. This Sequest analysis thus established that a high degree of sequence coverage was obtained for the protein, thus decreasing the possibility that detection of adducts was affected by poor sequence coverage. Several epoxide adducts were detected by SALSA analysis of the LC-MS-MS data (Table 1). Summaries of the b- and y-series ions for the target peptides and their modified forms are presented in Table 2. Modified variants of five Hbβ and two HbR tryptic peptides were detected in SALSA searches of the LC-MS-MS datafiles for analysis of tryptic digests of the alkylated hemoglobin mixture. In the Hbβ chain, amino acid targets included histidine (His 77, His 97, and His 143), Cys 93, and the N-terminal amine. In the HbR chain, modification was only observed at the N-terminal valine and at a single histidine residue (His 20). Incubation of hemoglobin with the epoxide mixture was repeated at epoxide concentrations of 40 µM. At the lower concentration, only the HbR and Hbβ N-terminal valine adducts and the Hbβ Cys 93 adducts of the epoxides were detected (data not shown). All of the epoxides modified the N-terminal valines of both Hb chains. As with the example above, the position of modification in each case was verified by inspection of the MS-MS spectra, in which the b-series ions all were displaced by the mass of the adduct. The y-series ions corresponded to those of the unmodified peptide. This verified the position of adduction at the N-terminus. Modification by styrene oxide and ethylene oxide corresponded to addition of the epoxide mass to the target

peptide. This corresponds mechanistically to nucleophilic attack at an epoxide carbon with ring opening to form a β-hydroxy-substituted adduct. Modification by butadiene dioxide produced two types of adducts that were distinguished by adduct mass. The first corresponded to a M + 86 adduct, which corresponds to either (1) addition of a nucleophile to one of the epoxides with ring opening to form a N-(2-hydroxy-3,4-epoxybutyl) adduct (butadiene dioxide adduct 1, Figure 1) or (2) internal cyclization of BDO adduct 1 by attack of the amine nitrogen on the remaining epoxide to form the N-(2,3-dihydroxy-1,4butyl) cyclic adduct (butadiene dioxide adduct 2, Figure 1). It is not possible to distinguish between these adducts with the instrumentation used in this study. The second type of adduct formed corresponded to a M + 104 adduct, which is a N-(2,3,4-trihydroxybutyl) adduct (butadiene dioxide adduct 3, Figure 1). This adduct arises from nucleophilic attack at one epoxide with hydrolytic ring opening of the other epoxide either before or after addition to the peptide. Detection of ethylene oxide adducts was complicated by the ubiquitous formation of N-terminal carbamyl adducts due to the urea used in workup of the proteins (see above). Because of the mass similarity of these modifications (43 amu for the carbamyl modification vs 44 amu for the ethylene oxide adduct), we could not reliably distinguish these modifications with the ion trap MS instrument used in this work. However, stable carbamylation modifications occur only at the N-termini of peptides and thus any such modifications observed at N-termini of peptides other than the N-terminal tryptic peptides were clearly ascribed to artifactual carbamylation during workup. Ethylene oxide modification at the valine N-termini of the N-terminal tryptic peptides was distinguished by different HPLC retention of the carbamylated peptides and the ethylene oxide adducts (data not shown).

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Table 2. MS-MS Spectral Features of Unmodified and Adduct-Modified Hb Peptides peptidea

modificationb

precursor m/z

b/y-ions detectedc 236.0 (b2), 349.0 (b3), 450.1 (b4), 676.3 (b6), 805.3 (b7) 147.0 (y1), 276.1 (y2), 406.2 (y3), 502.3 (y4), 603.3 (y5), 716.3 (y6) 220.2 (b1), 357.2 (b2), 470.2 (b3), 571.3 (b4), 797.3 (b6), 926.3 (b7), 1013.3 (b8) 276.1 (y2), 405.6 (y3), 502.2 (y4), 603.3 (y5), 716.3 (y6), 854.3 (y7) 280.2 (b2), 393.2 (b3), 494.3 (b4), 720.5 (b6), 849.4 (b7), 978.5 (b8) 405.6 (y3), 502.2 (y4), 603.3 (y5), 716.3 (y6), 853.5 (y7) 323.1 (b2), 436.1 (b3), 537.2 (b4), 635.4 (b5), 763.4 (b6), 892.5 (b7) 276.1 (y2), 405.3 (y3), 502.0 (y4), 603.4 (y5), 716.4 (y6), 853.5(y7) 341.1 (b2), 555.4 (b4), 781.8 (b6), 910.6 (b7) 276.1 (y2), 502.8 (y4), 715.9 (y6) 271.1 (b3), 374.2 (b4), 556.2* (b5), 690.4 (b6), 729.5* (b7), 860.3 (b8), 931.6 (b10), 1068.6 (b11), 1182.6 (b12), 1296.7 (b13), 1411.0 (b14), 1523.8 (b15) 260.2 (y2), 374.2 (y3), 489.3 (y4), 602.4 (y5), 739.4 (y6), 810.5 (y7), 923.8 (y8), 980.6 (y9), 1095.7 (y10), 1182.6 (y11), 1329.8 (y12), 1401.1 (y13), 1457.9 (y14), 1571.8 (y15) 372.5 (b4), 690.2 (b7), 860.5 (b9), 931.6 (b10), 1188.7 (b11), 1302.9 (b12), 1416.6 (b13), 1530.8 (b14), 1644.9 (b15) 374.3 (y3), 489.1 (y4), 602.4 (y5), 859.6 (y6), 930.6 (y7), 1043.5 (y8), 1100.7 (y9), 1215.7 (y10), 1302.9 (y11), 1449.9 (y12), 1521.2 (y13), 1577.8 (y14) 306.1 (b3), 377.1 (b4), 460.2* (b5), 573.3* (b6), 660.3* (b7), 789.3* (b8), 902.3* (b9), 1057.6 (b10), 1160.6 (b11), 1275.5 (b12) 262.0 (y2), 365.2 (y3), 502.2 (y4), 615.4 (y5), 744.4 (y6), 831.4 (y7), 944.5 (y8), 1045.5 (y9), 1116.4 (y10), 1263.7 (y11) 287.9* (b3), 377.1 (b4), 460.3* (b5), 573.2* (b6), 660.2* (b7), 789.4* (b8), 902.3* (b9), 1057.6 (b10), 1280.4 (b11), 1395.9 (b12) 262.4 (y2), 485.3 (y3), 622.4 (y4), 735.3 (y5), 864.5 (y6), 951.5 (y7), 1064.5 (y8), 1165.6 (y9), 1237.7 (y10), 1383.3 (y11) 288.5* (b3), 377.1 (b4), 460.1* (b5), 573.9 (b6), 807.5 (b8), 919.6 (b9), 1057.8 (b10), 1245.0 (b11), 1361.7 (b12) 262.1 (y2), 451.2 (y3), 589.3 (y4), 701.4 (y5), 830.5 (y6), 917.4 (y7), 1030.5 (y8), 1131.7 (y9), 1203.6 (y10), 1351.7 (y11) 287.8 (b3), 460.3* (b5), 676.7 (b7), 808.5 (b8), 902.4* (b9), 1057.8 (b10), 1265.8 (b11), 1380.6 (b12) 243.7* (y2), 469.4 (y3), 606.3 (y4), 719.4 (y5), 848.4 (y6), 935.5 (y7), 1048.6 (y8), 1149.7 (y9), 1220.9 (y10), 1350.2 (y11) 251.2 (b2), 350.3 (b3), 465.3 (b4), 691.4 (b6), 806.4 (b7), 953.4 (b8) 175.2 (y1), 322.3 (y2), 437.2 (y3), 547.0* (y4), 663.5 (y5), 778.4 (y6), 877.5 (y7), 1015.0 (y8) 251.3 (b2), 371.2 (b3), 470.3 (b4), 585.4 (b5), 682.5 (b6), 811.3 (b7), 925.5 (b8), 1073.6 (b9) 322.4 (y2), 436.4 (y3), 567.7 (y4), 662.5 (y5), 777.5 (y6), 876.6 (y7) 198.8 (b2), 270.1 (b3), 327.1 (b4), 426.2 (b5), 497.1 (b6), 610.9 (b7), 682.4 (b8), 778.2* (b9), 866.6 (b10), 1003.4 (b11) 284.2 (y2), 355.1 (y3), 468.4 (y4), 539.4 (y5), 653.4 (y6), 724.4 (y7), 823.5 (y8), 880.5 (y9), 951.6 (y10) 198.9 (b2), 270.0 (b3), 326.9 (b4), 426.2 (b5), 497.3 (b6), 611.4 (b7), 682.5 (b8), 795.1 (b9), 867.4 (b10), 1124.5 (b11) 404.2 (y2), 475.2 (y3), 588.2 (y4), 659.4 (y5), 773.4 (y6), 844.4 (y7), 943.3 (y8), 1000.5 (y9), 1071.6 (y10), 1170.4 (y11) 199.2 (b2), 270.3 (b3), 327.2 (b4), 426.1 (b5), 497.5 (b6), 611.2 (b7), 682.4 (b8), 795.5 (b9), 866.4 (b10), 1048.3 (b11) 328.4 (y2), 398.2 (y3), 511.3 (y4), 582.3 (y5), 696.4 (y6), 767.4 (y7), 849.1* (y8), 923.5 (y9), 994.6 (y10), 1093.5 (y11) 212.9 (b2), 300.2 (b3), 397.1 (b4), 468.2 (b5), 583.3 (b6), 711.4 (b7) 262.1 (y2), 333.2 (y3), 430.3 (y4), 517.3 (y5), 630.3 (y6) 333.1 (b2), 420.2 (b3), 517.2 (b4), 587.9 (b5), 703.3 (b6), 831.4 (b7) 262.2 (y2), 333.1 (y3), 430.3 (y4), 517.2 (y5), 630.2 (y6) 257.0 (b2), 343.2 (b3), 441.5 (b4), 494.2* (b5), 609.7* (b6), 755.4 (b7) 262.1 (y2), 333.2 (y3), 430.3 (y4), 517.3 (y5), 630.4 (y6) 365.1 (b4), 436.2 (b5), 493.3 (b6), 622.4 (b7), 785.4 (b8), 842.5 (b9), 913.5 (b10), 1042.5 (b11), 1113.5 (b12), 1227.7 (b13), 1355.5 (b14) 304.2 (y2), 418.3 (y3), 488.4 (y4), 617.4 (y5), 688.5 (y6), 745.5 (y7), 908.6 (y8), 1037.7 (y9), 1094.6 (y10), 1165.6 (y11), 1302.6 (y12), 1373.8 (y13) 485.3 (b4), 556.3 (b5), 613.5 (b6), 742.4 (b7), 905.5 (b8), 963.5 (b9), 1033.6 (b10), 1162.5 (b11), 1233.7 (b12), 1347.6 (b13), 1475.7 (b14) 286.8* (y2), 317.1 (y3), 488.3 (y4), 617.5 (y5), 688.0 (y6), 745.5 (y7), 908.6 (y8), 1037.6 (y9), 1094.6 (y10), 1165.7 (y11), 1404.5* (y12), 1493.7 (y13) 408.1 (b4), 479.3 (b5), 536.4 (b6), 665.3 (b7), 828.5 (b8), 885.3 (b9), 957.7 (b10), 1085.5 (b11), 1157.5 (b12), 1269.6 (b13), 1400.6 (b14) 305.3 (y2), 418.1 (y3), 488.4 (y4), 617.5 (y5), 687.9 (y6), 745.5 (y7), 908.6 (y8), 1037.8 (y9), 1094.7 (y10), 1165.6 (y11), 1399.8* (y13)

β(1-8)

unmodified

952.5

β(1-8)

+SO

537.2

β(1-8)

+EO

995.7

β(1-8)

+BDO1

520.1

β(1-8)

+BDO2

529.2

β(67-82)

unmodified

835.8

β(67-82)

+SO

896.4

β(83-95)

unmodified

711.4

β(83-95)

+SO

771.8

β(83-95)

+BDO1

754.6

β(83-95)

+BDO2

763.8

β(96-104)

unmodified

564.6

β(96-104)

+SO

624.2

β(133-144)

unmodified

575.7

β(133-144)

+SO

635.8

β(133-144)

+EO

597.1

R (1-7)

unmodified

729.42

R (1-7)

+SO

425.49

R (1-7)

+EO

774.46

R (17-31)

unmodified

765.6

R (17-31)

+SO

825.77

R (17-31)

+EO

787.3

a Hb chain and amino acid positions in the target peptide. b The modifications are SO, styrene oxide (M + 120); EO, ethylene oxide (M + 44); BDO1, butadiene dioxide adduct 1 or 3 (Figure 1) (M + 86); BDO2, butadiene dioxide adduct 2 (Figure 1) (M + 104). c Observed signals assigned as b- or y-ions are listed. Ions containing an adduct moiety are mass-shifted with respect to the corresponding ions in unmodified peptides and are listed in boldface. Ions annotated with an asterisk (*) reflect loss of H2O (18 amu).

804

Chem. Res. Toxicol., Vol. 15, No. 6, 2002

Discussion LC-tandem MS is the state of the art approach to peptide sequence analysis. Together with data mining tools such as Sequest, which automate the correlation of protein sequences with MS-MS spectra, LC-tandem MS permits the rapid, automated identification of proteins in complex samples (30, 31). The recent introduction of the SALSA algorithm allows the mining of LC-tandem MS data for spectra corresponding to specific peptides, as well as their modified and variant forms (13, 14). Here we have demonstrated that LC-tandem MS and SALSA can be applied to the identification and sequence-specific mapping of protein-xenobiotic adducts. This approach offers a means to identify new markers of exposure to chemicals and to probe mechanisms of chemically induced injury. The objective of this study was to apply LC-tandem MS and SALSA to map hemoglobin adducts of aliphatic epoxides at the amino acid sequence level. We employed three widely studied aliphatic epoxides, styrene oxide, ethylane oxide, and butadiene dioxide, which react with Hb to form adducts in vitro and in vivo. Previous studies have employed Edman degradation and GC-MS (18, 21, 22), Raney Ni reduction (19), and base-catalyzed hydrolysis (20, 32) to measure adducts to the HbR and Hbβ N-terminal valines, the Cys β-93 thiol and various carboxylic acids, respectively. These studies required different analytical methods to detect the different types of adducts. Another study employed FAB-MS-MS to map styrene oxide adducts to His R20 and His β143 in human hemoglobin (23). More recently, a combination of ESI and MALDI-TOF MS analyses mapped of butadiene monoxide to specific peptides within the mouse HbR and Hbβ polypeptide chains (24). The latter provided a singleassay approach to diffferent amino acid adducts, but did not provide definitive amino acid sequence location of the adducts. Our results indicate that LC-tandem MS and SALSA provide a powerful, generic approach to map different types of adducts at the level of amino acid sequence. Our approach to mapping xenobiotic adducts by LCMS-MS is to (1) digest proteins in the sample to peptides, (2) perform LC-MS-MS analysis to obtain MS-MS spectra of modified and unmodified peptides in the sample, (3) use sequence motif-based data analysis with SALSA to identify MS-MS spectra corresponding to both modified and unmodified variants of each target peptide, and (4) confirm of the position and mass of adducts by inspection of the highest ranked MS-MS spectra. It is important to point out in this context that SALSA is merely a tool to rank spectra based on their correspondence to ion series characteristic of the target sequences (14). SALSA scores are highly dependent on the specified search criteria for different peptide motifs and are useful only as a means to rank the MS-MS spectra in a datafile. A SALSA score itself does not establish the presence of an adduct; inspection of the indicated MS-MS scans is required to establish that the spectrum corresponds to a modified peptide. Search motifs for SALSA analyses of MS-MS data consist of a minimum of 5 and a maximum of 12 continuous amino acids (depending on the target peptide length) entered in the N-terminal to C-terminal direction. This establishes a “virtual ruler” that corresponds to the y-ion series (i.e., ions with charge localized on the

Badghisi and Liebler

C-terminal portion of the peptide) for MS-MS spectral comparisons. For these studies, search motifs all symmetrically spanned the central portion of each target peptide. The N-terminal and C-terminal amino acids in the peptide are not used as part of the search motif, as the corresponding ions generally are not found in the MSMS spectra. We have previously examined different search motif strategies for SALSA (14), and although the approach used here satisfactorily identified peptide MSMS spectra, other strategies (e.g., b-ion series searches, linked product ions) probably could be used with similar success. A recent paper by Moll et al. (24) described the application of ESI-MS to localizing butadiene monoxide adducts to specific peptides of mouse HbR and Hbβ. The ESI-MS analyses established the presence of multiple adducts on both globin chains isolated from mouse erythrocytes treated with the epoxide in vitro. Although the electrophile studied, the protein target (mouse Hb vs human Hb), and the exposure system were different from ours, important similarities were noted. First, a major site of adduction appeared to be the globin Nterminal peptides. Second, adduction was also noted in the Hbβ peptide containing Cys 93 (conserved in mouse and human Hbβ). Moll et al. inferred adduction at lysine residues based on the observation of incompletely cleaved peptides containing adducts and the apparent detection of lysine adducts in subsequent GC-MS analyses of acid hydrolysates. Similarly, adducted histidines, methionines, serines, and valines, but not cysteines, were also detected in the GC-MS analyses. They speculated that adduction at lysine may have been responsible for incomplete cleavages. Although this is certainly plausible, we note that incomplete cleavage is quite common in tryptic digests and that it is equally possible that the adducts were on other amino acids in any of the incompletely cleaved peptides. In contrast, we observed only N-terminal valine adducts, together with adduction of cysteine and histidine in specific sequences. An advantage of our approach is that the data provide unambiguous localization of adducts to specific amino acids within intact Hb peptides. SALSA-assisted analysis of LC-MS data by sequence motif searching is essentially a generic approach that can reveal any adduct that affects the MS-MS fragmentation of a peptide. The spectra are acquired by data-dependent scanning without regard to expected peptide adduct masses. Our previous work showed the detection of simple modifications (sulfur oxidation, cysteine desulfuration) through sequence motif analysis of MS-MS spectra of albumin (14). Here we have shown that this analytical approach is readily extended to map more chemically complex xenobiotic adducts in protein sequences. An important problem in assessment of environmental exposures is that real-world exposures often involve multiple agents. The approach we describe here would be applicable to the discovery of unknown or unanticipated protein adducts as markers of exposure. Thus, it is not necessary to anticipate the exact chemical structures or masses of adducts to detect them. Moreover, once the MS-MS spectra for the peptides in the sample are acquired, the data may be mined repeatedly to identify MS-MS spectra that display different features of interest. In this way, a single LC-MS-MS analysis of a protein sample can be used to probe for different xenobiotic

Sequence Mapping Hemoglobin Adducts

adducts, endogenous posttranslational modifications, or any other characteristic that is manifested in MS-MS spectra.

Acknowledgment. This work was supported in part by NIH Grants ES10056 and ES06694.

References (1) Pandey, A., and Mann, M. (2000) Proteomics to study genes and genomes. Nature 405, 837-846. (2) Yates, J. R. (2000) Mass spectrometry. From genomics to proteomics. Trends Genet. 16, 5-8. (3) Cohen, S. D., Pumford, N. R., Khairallah, E. A., Boekelheide, K., Pohl, L. R., Amouzadeh, H. R., and Hinson, J. A. (1997) Selective protein covalent binding and target organ toxicity. Toxicol. Appl. Pharmacol. 143, 1-12. (4) Nelson, S. D., and Pearson, P. G. (1990) Covalent and noncovalent interactions in acute lethal cell injury caused by chemicals. Annu. Rev. Pharmacol. Toxicol. 30, 169-195. (5) Skipper, P. L., Obiedzinski, M. W., Tannenbaum, S. R., Miller, D. W., Mitchum, R. K., and Kadlubar, F. F. (1985) Identification of the major serum albumin adduct formed by 4-aminobiphenyl in vivo in rats. Cancer Res. 45, 5122-5127. (6) Jiao, K., Mandapati, S., Skipper, P. L., Tannenbaum, S. R., and Wishnok, J. S. (2001) Site-selective nitration of tyrosine in human serum albumin by peroxynitrite. Anal. Biochem. 293, 43-52. (7) Qiu, Y., Burlingame, A. L., and Benet, L. Z. (1998) Mechanisms for covalent binding of benoxaprofen glucuronide to human serum albumin. Studies By tandem mass spectrometry. Drug. Metab. Dispos. 26, 246-256. (8) Ding, A., Ojingwa, J. C., McDonagh, A. F., Burlingame, A. L., and Benet, L. Z. (1993) Evidence for covalent binding of acyl glucuronides to serum albumin via an imine mechanism as revealed by tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S.A 90, 3797-3801. (9) Kaur, S., Hollander, D., Haas, R., and Burlingame, A. L. (1989) Characterization of structural xenobiotic modifications in proteins by high sensitivity tandem mass spectrometry. Human hemoglobin treated in vitro with styrene 7,8-oxide. J. Biol. Chem. 264, 16981-16984. (10) Bolgar, M. S., Yang, C. Y., and Gaskell, S. J. (1996) First direct evidence for lipid/protein conjugation in oxidized human lowdensity lipoprotein. J. Biol. Chem. 271, 27999-28001. (11) Erve, J. C., Barofsky, E., Barofsky, D. F., Deinzer, M. L., and Reed, D. J. (1995) Alkylation of Escherichia coli thioredoxin by S-(2chloroethyl)glutathione and identification of the adduct on the active site cysteine-32 by mass spectrometry. Chem. Res Toxicol. 8, 934-941. (12) Yates, J. R., Eng, J. K., McCormack, A. L., and Schieltz, D. (1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426-1436. (13) Hansen, B. T., Jones, J. A., Mason, D. E., and Liebler, D. C. (2001) SALSA: a pattern recognition algorithm to detect electrophileadducted peptides by automated evaluation of CID spectra in LCMS-MS analyses. Anal. Chem. 73, 1676-1683. (14) Liebler, D. C., Hansen, B. T., Davey, S. W., Tiscareno, L., and Mason, D. E. (2001) Peptide sequence motif analysis of tandem MS data with the SALSA algorithm. Anal. Chem. (in press). (15) Skipper, P. L., Peng, X., Soohoo, C. K., and Tannenbaum, S. R. (1994) Protein adducts as biomarkers of human carcinogen exposure. Drug Metab. Rev. 26, 111-124.

Chem. Res. Toxicol., Vol. 15, No. 6, 2002 805 (16) Farmer, P. B. (1995) Monitoring of human exposure to carcinogens through DNA and protein adduct determination. Toxicol. Lett. 82-83, 757-762. (17) Ehrenberg, L., Granath, F., and Tornqvist, M. (1996) Macromolecule adducts as biomarkers of exposure to environmental mutagens in human populations. Environ. Health Perspect. 104 (Suppl. 3), 423-428. (18) Sepai, O., Anderson, D., Street, B., Bird, I., Farmer, P. B., and Bailey, E. (1993) Monitoring of exposure to styrene oxide by GCMS analysis of phenylhydroxyethyl esters in hemoglobin. Arch. Toxicol. 67, 28-33. (19) Rappaport, S. M., Ting, D., Jin, Z., Yeowell-O’Connell, K., Waidyanatha, S., and McDonald, T. (1993) Application of Raney nickel to measure adducts of styrene oxide with hemoglobin and albumin. Chem. Res. Toxicol. 6, 238-244. (20) Yeowell-O’Connell, K., Jin, Z., and Rappaport, S. M. (1996) Determination of albumin and hemoglobin adducts in workers exposed to styrene and styrene oxide. Cancer Epidemiol. Biomarkers Prev. 5, 205-215. (21) Tornqvist, M., Mowrer, J., Jensen, S., and Ehrenberg, L. (1986) Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method. Anal. Biochem. 154, 255-266. (22) Pauwels, W., Farmer, P. B., Osterman-Golkar, S., Severi, M., Cordero, R., Bailey, E., and Veulemans, H. (1997) Ring test for the determination of N-terminal valine adducts of styrene 7,8oxide with haemoglobin by the modified Edman degradation technique. J. Chromatogr. B 702, 77-83. (23) Kaur, S., Hollander, D., Haas, R., and Burlingame, A. L. (1989) Characterization of structural xenobiotic modifications in proteins by high sensitivity tandem mass spectrometry. Human hemoglobin treated in vitro with styrene 7,8-oxide. J. Biol. Chem. 264, 16981-16984. (24) Moll, T. S., Harms, A. C., and Elfarra, A. A. (2000) A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide. Chem. Res. Toxicol. 13, 1103-1113. (25) Jones, J. A., and Liebler, D. C. (2000) Tandem MS analysis of model peptide adducts from reactive metabolites of the hepatotoxin 1,1-dichloroethylene. Chem. Res Toxicol. 13, 1302-1312. (26) Mason, D. E., and Liebler, D. C. (2000) Characterization of benzoquinone-peptide adducts by electrospray mass spectrometry [In Process Citation]. Chem. Res Toxicol. 13, 976-982. (27) Harriman, S. P., Hill, J. A., Tannenbaum, S. R., and Wishnok, J. S. (1998) Detection and identification of carcinogen-peptide adducts by nanoelectrospray tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 9, 202-207. (28) Stark, G. R. (1965) Reactions of cyanate with functional groups of proteins. III. Reactions with amino and carboxyl groups. Biochemistry 4, 1030-1036. (29) Stark, G. R., Stein, W. H., and Moore, S. (1960) Reactions of cyanate present in aqueous urea with amino acids and proteins. J. Biol. Chem. 235, 3177-3181. (30) Yates, J. R., III (1998) Mass spectrometry and the age of the proteome. J. Mass Spectrom. 33, 1-19. (31) Yates, J. R. (2000) Mass spectrometry. From genomics to proteomics. Trends Genet. 16, 5-8. (32) Yeowell-O’Connell, K., Pauwels, W., Severi, M., Jin, Z., Walker, M. R., Rappaport, S. M., and Veulemans, H. (1997) Comparison of styrene-7,8-oxide adducts formed via reaction with cysteine, N-terminal valine and carboxylic acid residues in human, mouse and rat hemoglobin. Chem.-Biol. Interact. 106, 67-85.

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