Chem. Res. Toxicol. 1996, 9, 781-787
781
Characterization of Sulfur Mustard Induced Structural Modifications in Human Hemoglobin by Liquid Chromatography-Tandem Mass Spectrometry Daan Noort,*,† Elwin R. Verheij,‡ Albert G. Hulst,§ Leo P. A. de Jong,† and Hendrik P. Benschop† Department of Chemical Toxicology and Department of Analysis of Toxic and Explosive Substances, TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands, and Center for Structure Elucidation and Instrumental Analysis, TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received December 29, 1995X
In this paper we describe the use of tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to bis(2-chloroethyl) sulfide (sulfur mustard). Globin isolated from human whole blood which had been exposed to sulfur mustard was degraded with trypsin, and the digests were analyzed by micro LC/MS. Alkylated tryptic fragments (R-T1, R-T4, R-T6, R-T9, β-T1, β-T9, β-T10, β-T11, and β-T10-S-S-β-T12) could be tentatively assigned upon comparison with a digest from nonexposed globin. Subsequent tandem mass spectrometry of these peptides allowed unambiguous assignment of 5 specific modified residues: R-Val-1, R-His-20, β-Val-1, β-His-77, and β-His-97. The results demonstrate the usefulness of microbore LC in combination with tandem mass spectrometry for the structural determination of chemically modified peptides and proteins.
Introduction The development of analytical methods to detect the nature and extent of poisoning with chemical warfare agents has gained serious attention due to the proliferation and use of such agents in warfare and in terroristic attacks in recent years (1). Within this context, we are engaged in the development of methods for retrospective detection of exposure to bis(2-chloroethyl) sulfide (sulfur mustard; 1, see Scheme 1). Sulfur mustard is a primary carcinogenic, cytotoxic, and strongly vesicant agent which has been used as a chemical warfare agent since World War I (2). The high reactivity of sulfur mustard in aqueous media is thought to be mediated by intramolecular displacement of one of the β-chlorine atoms by the sulfur atom, giving rise to a highly reactive electrophilic episulfonium ion 2. Subsequent reaction with a nucleophile followed by hydrolysis of the second mustard functionality will yield adducts 3, although the formation of diadducts has also been observed, e.g., for glutathione (3) and for (closely spaced) deoxyguanosine residues in DNA (4). The in vivo metabolism (in rats) of sulfur mustard has been studied in detail by Black and co-workers (3). Due to presence of two electrophilic sites and due to oxidation of sulfur, a wide array of metabolites results, most of which are rapidly excreted into the urine. For use in detection of alleged exposure to chemical agents, it should be taken into consideration that biological samples often * To whom correspondence should be addressed, at the TNO Prins Maurits Laboratory, P.O. Box 45, 2280 AA Rijswijk, The Netherlands. Telephone: +31 15 284 34 97; telefax: +31 15 284 39 63. † Department of Chemical Toxicology, TNO Prins Maurits Laboratory, Rijswijk. ‡ Center for Structure Elucidation and Instrumental Analysis, TNO Nutrition and Food Research, Zeist. § Department of Analysis of Toxic and Explosive Substances, TNO Prins Maurits Laboratory, Rijswijk. X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0893-228x(95)00214-1 CCC: $12.00
Scheme 1
become available only days or even weeks after exposure. Therefore, we have chosen to assess exposure to sulfur mustard by analysis of adducts to DNA and proteins. In previous papers we described the synthesis, characterization, and quantitation of the major adducts formed between sulfur mustard and DNA (4) and the development of a sensitive ELISA for the major DNA adduct of sulfur mustard, i.e., 2′-deoxy-N7-[2-[(hydroxyethyl)thio]ethyl]guanosine (5). We now focus our attention toward mass spectrometric and immunochemical detection of hemoglobin-sulfur mustard adducts. Since hemoglobin is much more abundant than DNA, it can be expected that more adducts of hemoglobin result, which may facilitate analysis. Moreover, hemoglobin adducts have a longer life span (∼120 days) than DNA adducts, which are subject to enzymatic repair. Mass spectrometry has been employed extensively in the characterization of adducts of proteins with xenobiotics (6). Cleavage of N-terminal valine adducts in hemoglobin by reaction with pentafluorophenyl isothiocyanate and their subsequent analysis by GC/MS has been reported for numerous electrophilic agents (7) and is the subject of our succeeding paper concerned with valine-sulfur mustard adducts (8). Other approaches involve acidic hydrolysis or nonspecific enzymatic digestion of adducted proteins, followed by derivatization and GC/MS analysis of modified amino acids (9). Following these approaches, however, specific modified sequences in the proteins cannot be revealed. For the unambiguous determination of modified sites within proteins, recently developed tandem mass spectrometric techniques can be applied. For instance, Kaur et al. (10) identified the sites © 1996 American Chemical Society
782 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
of alkylation of styrene oxide after tryptic digestion of human hemoglobin treated with styrene oxide, by applying tandem mass spectrometric analysis of the resulting tryptic fragments. Following a similar approach, Ding et al. (11, 12) elucidated the binding sites of tolmetin glucuronide with human serum albumin, whereas Erve et al. identified the adducts of S-(2-chloroethyl)glutathione with oxytocin (13) and thioredoxin (14). Recently, Fenselau and co-workers (15) reported on the use of tandem mass spectrometry to elucidate structural modifications in metallothionein upon alkylation by melphalan. In this paper we report on the identification of covalent modifications in human hemoglobin after exposure to sulfur mustard, using tandem mass spectrometry hybridized with micro-HPLC. Identification of these alkylation sites will form the basis for selecting peptide fragments which can be used as haptens in future immunoassay development.
Materials and Methods Chemicals. Caution: Sulfur mustard is a primary carcinogenic, vesicant, and cytotoxic agent. This compound should be handled only in fume cupboards by experienced personnel. Human blood was obtained from healthy volunteers, with consent of the donor and approval of the TNO Medical Ethical Committee. Technical-grade sulfur mustard was distilled before use to a gas chromatographic purity exceeding 99.5%. For the synthesis of [35S]sulfur mustard, hydrogen [35S]sulfide was obtained by reduction of sodium [35S]sulfate (carrier free; 0.9 GBq; 44-52 TBq/mmol; Dupont du Nemours) with HI/HCOOH/ sodium hypophosphite (16). This was subsequently reacted with ethylene oxide followed by chlorination of the formed [35S]thiodiglycol with thionyl chloride, as described by Boursnell et al. (17). Immobilized TPCK-trypsin (>45 units/mL of gel) was obtained from Pierce (Rockford, IL). Instrumentation. HPLC analyses of [35S]-labeled tryptic digests were carried out on a PepRPC 5/5 column (Pharmacia, Uppsala, Sweden) using two Waters 510 HPLC pumps and an Applied Biosystems 757 detector connected to a Radiometric Floone/Beta A-500 radiochromatography detector (Canberra Packard, Tilburg, The Netherlands) with Flo-Scint A (Canberra Packard) as a scintillation cocktail. Semipreparative FPLC was performed on a Pharmacia FPLC system (consisting of 2 P-500 pumps, an LCC-501 controller, and a UV-M II monitor) using a PepRPC 10/10 column. Electrospray LC/MS and collisionally induced dissociation (CID)1 tandem MS analyses of tryptic digests were performed on a Finnigan MAT TSQ700 triple quadrupole mass spectrometer (Finnigan MAT, Hemel Hempstead, U.K.) equipped with an API LC/MS interface and an ESP2 electrospray source. The solvent delivery apparatus in the LC system consisted of a Waters 600-MS quaternary gradient pump. Electrospray MS analysis of globin chains was performed on a VG Quattro II triple quadrupole mass spectrometer (Fisons, Altrincham, U.K.). Exposure of Human Blood to Sulfur Mustard or [35S]Sulfur Mustard. Fresh heparinized blood (20 mL) was incubated with sulfur mustard (25 mM) or [35S]sulfur mustard (0.31 GBq/mmol; 5 mM) by addition of a solution of the agent in CH3CN (200 µL) followed by incubation for 1 h at 37 °C. Erythrocytes were isolated by centrifugation for 10 min at 3000 rpm. After removal of plasma the erythrocytes were washed with saline (4×) and lysed with H2O. After 30 min at 4 °C, the hemolysate was centrifuged for 30 min at 25000g (4 °C). The supernatant was dialyzed overnight against H2O. Globin was isolated according to the method described by Bailey et al. (18). Tryptic Cleavage of Globin from Blood Exposed to [35S]Sulfur Mustard and Subsequent HPLC Analysis. Globin 1 Abbreviations: CID, collisionally induced dissociation; TFA, trifluoroacetic acid.
Noort et al. (3 mg) isolated from blood exposed to [35S]sulfur mustard (5 mM; sp act. 8.4 mCi/mmol) was dissolved in H2O (0.5 mL). Immobilized TPCK-treated trypsin [0.25 mL gel; suspended in aqueous NH4HCO3 (0.5 mL; 0.1 M; pH 8.0)] which had been washed with aqueous NH4HCO3 (5 × 4 mL; 0.1 M; pH 8.0) was added. The mixture was incubated at 37 °C for 16 h in a shaking device. Subsequently, an additional batch of trypsin was added [0.25 mL; suspended in aqueous NH4HCO3 (0.5 mL; 0.1 M; pH 8.0)], and the mixture was incubated for another 16 h. The mixture was filtered through a Millex HV 13 filter to remove the immobilized trypsin and precipitated protein. The clear solution was concentrated under reduced pressure and redissolved in 0.1% trifluoroacetic acid (TFA)/H2O (200 µL). Reversed phase HPLC analysis was performed with UV detection (214 nm) combined with radiometric detection. A linear gradient (0-25 min; flow rate 1.0 mL/min) was applied from 0.1% TFA in H2O to 0.1% TFA in CH3CN/H2O (64/36 v/v) followed by elution with 0.1% TFA in CH3CN/H2O (80/20 v/v) during 5 min. Injection volume: 25 µL. LC/MS and Tandem MS Analyses of Tryptic Digests. Microcolumns were prepared from glass-lined stainless steel tubing (i.d. 0.32 mm; length 250 mm) with Spherisorb ODS2 S5 (PhaseSep) as the stationary phase. The LC pump operated at 1 mL/min. A preinjector split was made by the parallel combination of the microcolumn and an Adsorbosphere C18 column (Alltech; 150 × 3 mm; 5 µm particles). The split ratio was approximately 1:200, resulting in a flow rate of approximately 5 µL/min through the microcolumn, based on the observed dead time of the system. Injections were done using a Valco six-port injector equipped with a 10 µL loop. The mobile phases consisted of (A) 0.05% TFA in H2O and (B) 0.05% TFA in CH3CN/H2O, 80/20 (v/v). The applied modifier gradient was as follows: 0-5 min: 100% A; 5-35 min: 100% A to 100% B, linear; 35-40 min: 100% B; 41-45 min: 100% A. The operating conditions of the electrospray interface were as follows: voltage 5 kV, sheath gas setting 40, and a heated capillary temperature of 200 °C. For tandem MS experiments argon was used as the collision gas at pressures of 0.3-0.4 Pa, and the collision energy was 20-40 V. The parent resolution was slightly decreased to improve the sensitivity.
Results Quantitation of Covalent Binding of Sulfur Mustard to Hemoglobin. Electrospray MS analysis of globin isolated from human blood after exposure to sulfur mustard (25 mM) clearly demonstrated that hemoglobin is efficiently alkylated by sulfur mustard (see Figure 1). Multiply charged ions for the native R- and β-chains as well as for the mono- and dialkylated globin chains were detected; a true mass scale spectrum was obtained after transformation (19) of the multiply charged ions by application of a maximum entropy (MaxEnt) algorithm that enhances resolution and noise-to-noise ratios. From the transformed spectrum, Figure 1(b), showing the mass increments of 104 Da corresponding with the 2-[(hydroxyethyl)thio]ethyl moiety, it can be estimated that the degree of modification of globin is 30-40%, i.e., an average of about 1 modified chain versus 2 native chains. If an approximately linear relationship between exposure level and alkylation degree is assumed, this result is roughly consistent with the degrees of modification found for globin isolated from blood that was treated with [35S]sulfur mustard at lower concentrations, i.e., 13% (5 mM) and 2% (1 mM) (8). Tryptic digests of globin isolated from human blood that had been exposed to [35S]sulfur mustard (5 mM) gave reproducible reversed phase HPLC chromatograms (see Figure 2). The recovery of radioactivity after tryptic digestion and workup was 43%. Four major radioactive peaks could be detected. The first peak probably contains
Tandem MS of Hemoglobin-Sulfur Mustard Adducts
Figure 1. Electrospray mass spectrometry of globin isolated from human blood after exposure to sulfur mustard (25 mM). (a) Mass spectrum of multiply charged ions of native R-chain (A) and β-chain (B), monoalkylated R-chain (C) and β-chain (D), and dialkylated R-chain (E) and β-chain (F); the digits denote the number of charges. (b) Reconstructed MaxEnt spectrum obtained by transformation of multiply charged ions to an average mass, represented on a real mass scale; calculated molecular masses for the native R- and β-chains of globin are 15126.38 and 15867.24, respectively.
Figure 2. HPLC analysis of tryptic digests of globin isolated from human blood exposed to [35S]sulfur mustard (5 mM). (a) Radiometric detection; (b) UV detection.
small peptides and/or thiodiglycol released from glutamic acid and aspartic acid adducts. The second peak in the HPLC radiochromatogram corresponds with 1/8 of the total radioactivity in the analyzed sample. When it is assumed that the peak represents one single type of tryptic fragment, it follows that the degree of modification of this particular native tryptic fragment is 1.6%, i.e., (1/8) × 13%. Micro-LC/MS Analyses of Tryptic Fragments. Trypsinized globin samples from blood exposed to sulfur mustard (25 mM) or from nonexposed blood were used for micro-LC/MS analyses. In order to facilitate mass spectrometric analysis of tryptic digests, R- and β-chains of globin were separated prior to trypsin treatment, according to a published procedure (20). Initially, we directed our attention toward the mass spectrometric characterization of alkylated tryptic fragments of β-globin. Hydrophilic peptides were not retained on the column, and consequently, data are not available for peptides β-T6, β-T7, β-T8, and β-T15. To investigate the presence of alkylated peptides, chromatograms for tryptic frag-
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 783
ments of exposed and nonexposed globin samples were compared and systematically searched for m/z differences of 104, 52, and 34.7 (for [M + H]+, [M + 2H]2+, and [M + 3H]3+ ions, respectively). This procedure resulted in the detection of (possibly) alkylated β-T1, β-T9, β-T10, β-T11, and β-T10-S-S-β-T12 (see Table 1). The chromatograms for β-T1 are dealt with in detail, as an example (see Figure 3). The chromatograms contain the traces m/z 477 and 952.7 for the [M + 2H]2+ and [M + H]+ ions of the native peptide and the traces m/z 529 and 1056.7 for the [MA + 2H]2+ and [MA + H]+ ions of the alkylated peptide. When the chromatograms for the two samples are compared, a clear additional peak (retention time 22.4 min) is observed for the sulfur mustard-exposed sample (retention time native tryptic fragment 21.6 min). To identify an additional peak as an alkylated product, it is essential that the peak be observed in both the [MA + H]+ and the [MA + 2H]2+ traces (or other combinations if available) at the same retention time. Due to the extremely low solubility of purified R-globin in the buffer used for tryptic cleavage, it was decided to use crude trypsinized R/β-globin for analysis of alkylated R-tryptic fragments. The samples were analyzed for alkylated tryptic fragments as described above, which resulted in the detection of (possibly) alkylated R-T1, R-T4, R-T6, and R-T9 (see Table 2). Data for R-T2, R-T7, R-T8, R-T10, and R-T14 are not available, due to the hydrophilicity of these peptides. It was attempted to relate alkylated tryptic fragments found by electrospray LC/MS analyses to radioactive peaks observed in the HPLC chromatogram of a digest of globin isolated from [35S]sulfur mustard-exposed human blood. For this purpose, a tryptic digest of globin isolated from human blood that had been exposed to 5 mM sulfur mustard was fractionated by semipreparative reversed phase FPLC and the fractions were analyzed by electrospray LC/MS after concentration. Unfortunately, only the corresponding native fragments could be detected, which is probably caused by the relatively low exposure level. However, since we found earlier that the retention times for nonalkylated peptides and the corresponding alkylated peptide differ only slightly, the following tentative assignments can be made. Both R-T1 and β-T1 elute in the region of the chromatogram for globin digest in which the UV-absorbing peaks 5-7 were found (see Figure 2), whereas the peptides R-T4, β-T11, and β-T9 elute in the regions for peaks 10-12, for peaks 15 and 16, and for peak 17, respectively. The peptides β-T1 and β-T11 may coincide with the third and the small fifth radioactive peak, respectively. Distinct radioactive peaks were not found in the region of the chromatogram where β-T9 elutes, suggesting that the concentration of the corresponding alkylated peptide in the digest is low. The other peptides (R-T6, R-T9, β-T10, and β-T10S-S-β-T12) could no longer be detected after fraction collection. Sequencing of Alkylated Tryptic Fragments by CID Tandem MS Analysis. The results presented thus far only provide molecular mass information. CID tandem MS experiments are required to determine the site of alkylation. Satisfactory CID tandem MS spectra were obtained for alkylated R-T1, R-T4, β-T1, β-T9, and β-T11. For R-T1 (Val1-Leu-Ser-Pro-Ala-Asp-Lys7) the mass spectrum (for MH+) of the native peptide contains inter alia the full set of Y′′ ions (for nomenclature, see ref 21) and an abundant A2 ion (spectrum not shown). The B3
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Noort et al.
Table 1. HPLC Retention Times and m/z Values of Peptides Originating from the β-Chain of Globin Found by LC/MS Analysis of a Tryptic Digest of β-Globin Isolated from Nonexposed Human Blood (M) and from Human Blood after Exposure to 25 mM Sulfur Mustard (MA) peptide
retention time (min)
[M + H]+
[M + 2H]2+
β-T1 β-T2 β-T3 β-T4 β-T5 β-T9 β-T10 β-T11 β-T12 β-T13 β-T14 β-T10-S-S-T12
21.6/22.4a
952.7 932.6 1314.7 1275.0
477.0 467.0 658.5 638.2 1030.3 836.1 711.8 564.1 860.5 690.4 575.5 1570.5
a
26.3 24.7 29.7 27.9 29.0/30.8a 25.8/26.0a 24.5/25.5a 42.8 23.4 23.7 29.5/29.5a
1670.0 1422.0 1126.5 1379.0 1149.7
[M + 3H]3+
687.3 558.3
[MA + H]+
[MA + 2H]2+
1056.7
529.0
1774.0 1526.0 1230.5
888.1 763.8 616.3
574.6
[MA + 3H]3+
593.0 609.3
1047.5
1622.5
1082
Retention time of alkylated sequence.
Figure 3. Electrospray LC/MS chromatograms for β-T1 (Val1-His-Leu-Thr-Pro-Glu-Glu-Lys8) in a tryptic digest of native human globin (a) and of globin isolated from human blood after exposure to sulfur mustard (25 mM) (b) The peaks arising from the native peptide are marked with “N” and those of the candidate alkylated peptide with “N*”. Table 2. HPLC Retention Times and m/z Values of Peptides Originating from the r-Chain of Globin Found by LC/MS Analysis of a Tryptic Digest of Globin Isolated from Nonexposed Human Blood (M) and from Human Blood after Exposure to 25 mM Sulfur Mustard (MA) peptide R-T1 R-T3 R-T4 R-T5 R-T6 R-T9 R-T11 R-T12 R-T13 a
retention times (min)
[M + H]+
[M + 2H]2+
20.5/22.2a
729.4 532.3 1529.7 1071.6
22.3 23.2/24.0a 27.7 26.7/27.5a 30.7/31.8a 23.5 36.7 27.0
818.6 1252.7
[M + 3H]3+
[MA + H]+
[MA + 2H]2+
365.2
833.4
417.2
765.5 536.5 918.1 1499.7 409.9 1484.8 627.3
1633.7
817.5
612.4 1000.1
969.6 1551.7
[MA + 3H]3+
647.1 1034.4
990.3
Retention time of alkylated sequence.
ion at m/z 404 in the spectrum of the alkylated peptide (see Figure 4) is consistent with a modification at one of the first three amino acids. The A1 ion at m/z 176 indicates that the terminal valine has been modified (HOCH2CH2SCH2CH2NH-valine). In addition, the spec-
trum contains the Y′′, Y5′′, and Y6′′ ions at the same m/z as that of the native peptide, which is further proof for alkylation at the valine residue. The characteristic ion (R) observed at m/z 105 is ascribed to the adduct fragment [CH2CH2SCH2CH2OH]+.
Tandem MS of Hemoglobin-Sulfur Mustard Adducts
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 785
Figure 4. CID tandem MS spectrum for molecular ion [MA + H]+ (m/z 833) of alkylated R-T1. R ) [CH2CH2SCH2CH2OH]+.
Figure 6. CID tandem MS spectrum for molecular ion [MA + 2H]2+ (m/z 888) of alkylated β-T9.
Figure 5. CID tandem MS spectrum for molecular ion [MA + 2H]2+ (m/z 817) of alkylated R-T4. R ) [CH2CH2SCH2CH2OH]+; I* ) HOCH2CH2SCH2CH2-imidazolyl-CH2CHdN+H2.
In the case of R-T4 (Val17-Gly-Ala-His-Ala-Gly-Glu-TyrGly-Ala-Glu-Ala-Leu-Glu-Arg31), the mass spectrum (for MH22+) of the native peptide contains a number of Y′′ fragments, some other sequence ions, e.g., a strong A4 ion at m/z 337, and a fragment at m/z 110 which is probably the immonium ion of histidine: imidazolyl-CH2CHdN+H2 (spectrum not shown). The mass spectrum of the alkylated peptide (see Figure 5) is not of a very good quality because native R-T11 eluted in the close proximity of alkylated R-T4. The difference in m/z between the doubly charged ion of alkylated R-T4 and the [M + H]+ ion of R-T11 is only 1, which indicates that the tandem MS spectrum may contain some fragment ions from R-T11. Nevertheless, the presence of m/z 105 (R ) [CH2CH2SCH2CH2OH]+) and m/z 1530-1531 ([MA + H - 104]+, similar to MH+ of the native peptide)
indicates that this peptide is indeed alkylated R-T4. The histidine immonium ion at m/z 110 is not present; the fragment at m/z 214 (104 shift) is probably the immonium ion of alkylated histidine. This suggests that the alkylation site is histidine. Further proof for this is the absence of the A4 ion at m/z 337 and the presence of A4, B4, A5, and B5 fragments at m/z 441, 469, 512, and 540, respectively. The Y′′ series of ions is of low intensity and is up to Y10′′ or Y11′′ at the same m/z values as those for the native peptide, indicating that the modification is in the first 4 or 5 N-terminal amino acids. This finding is in agreement with alkylation at His-20. For β-T1 (Val1-His-Leu-Thr-Pro-Glu-Glu-Lys8), the spectra (for MH+) of the native and the alkylated peptide contain Y2′′, Y3′′, Y4′′, Y5′′, Y6′′, and Y7′′ ions at the same m/z values, as was the case for native and alkylated R-T1 (spectra not shown). This implies that the modification is at amino acid 1. The ion at m/z 176 for the alkylated peptide is in agreement with an A1 ion for alkylated N-terminal valine, as was the case for alkylated R-T1. The fragment B2 at m/z 237 for the native peptide shifts to a fragment at m/z 341 for the alkylated peptide, which is indicative for modification at valine or histidine. Furthermore, the characteristic ion at m/z 105 (R ) [CH2CH2SCH2CH2OH]+) and the [MA + H - 104]+ ion at m/z 952 (similar to [M + H]+ of native β-Τ1) are present. The spectrum (for MH22+) of alkylated β-T9 (Val67-LeuGly-Ala-Phe-Ser-Asp-Gly-Leu-Ala-His-Leu-Asp-Asn-LeuLys82) is given in Figure 6. Since a number of the lower (e4) Y′′ fragments in the alkylated and native peptide have the same m/z value and a number of the higher (g7) Y′′ fragments show a m/z difference of 104, alkylation has probably occurred at either His-77 or at Leu78. Since the side chain function of leucine cannot be alkylated, these data indicate that alkylation occurred at His-77. A small signal at m/z 1173 in the spectrum of the alkylated peptide, which may be the B11 ion, supports this hypothesis. Unfortunately, the B10 ion is not observed. The signal at m/z 1286-1287 in the
786 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Figure 7. CID tandem MS spectrum for molecular ion [MA + 2H]2+ (m/z 616) of alkylated β-T11. R ) [CH2CH2SCH2CH2OH]+.
spectrum of the alkylated peptide might be due to B12 and/or Y11′′ (calculated monoisotopic mass for alkylated B12 is 1285.6, and for alkylated Y11” 1286.6). For β-T11 (Leu96-His-Val-Asp-Pro-Glu-Asn-Phe-Arg104), the spectra for both the native and the alkylated peptide (for MH22+) contain the Y1′′ to Y7′′ ions (with the exception of Y4”) at the same m/z values, which indicates that alkylation occurred at either Leu-96 or His-97 (Figure 7). Further proof for this is found from the shift of 104 in the B2 fragment from m/z 251 to 355. The characteristic ion at m/z 105 (R ) [CH2CH2SCH2CH2OH]+) and the [MA + H - 104]+ ion are also observed for this alkylated peptide. Since the side chain function of leucine cannot be alkylated, it can be concluded that the alkylation of β-T11 occurred at His-97.
Discussion In this paper, the use of LC-tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to sulfur mustard is described. Initial evidence for covalent binding of sulfur mustard to hemoglobin was obtained from electrospray ionization MS experiments. This technique (22) allowed the accurate mass measurement of globin chains modified by the agent. The results show that hemoglobin is efficiently alkylated by sulfur mustard since approximately 11-14% of the administered sulfur mustard is covalently attached to the protein (globin concentration in blood: 9 mM). Recently, this approach has been reported for the determination of globin adducts of acrylamide (23), of 4-hydroxy-2-nonenal (24), and of methyl bromide (25). Information on the sites of alkylation within the tertiary structure of hemoglobin was obtained by the use of tandem mass spectrometry hybridized with micro-LC. The on-line analysis of complex mixtures by combined liquid chromatography-mass spectrometry is superior to off-line analysis of fractions collected after chromatographic separation, as has been performed by others (1015). For instance, the presence of trace amounts of alkylated peptides may be obscured by the highly abundant native peptides, as was already mentioned by Kaur
Noort et al.
et al. (10). In addition, more labile adducts might (partly) decompose during the isolation procedure. Furthermore, background subtraction procedures are less effective in the mass spectrometric analysis of fractions than in online mass spectrometric procedures. Fraction collection is also laborious and results in a substantial increase in the number of samples to be analyzed by MS. In spite of the large injection volume (10 µL) relative to the column dimensions, a large number of distinct peaks could be observed. In electrospray mass spectrometry, the formation of ions is supposed to be a concentration dependent process, as opposed to mass flow sensitive MS techniques. Since the analyte concentration in the column effluent is proportional to (i.d.)-2, a reduction of i.d. is advantageous for improving the detection limit (26). In these investigations, micro-LC columns with an i.d. of 0.32 mm were used, after observing that columns with an i.d. of 2.0 mm did not result in the required sensitivity. Evidence was obtained for the presence of alkylated β-T1, β-T9, β-T10, β-T11, β-T10-S-S-β-T12, R-T1, R-T4, R-T6, and R-T9. The slight increase in retention time observed for the alkylated peptides is in accordance with the increase in hydrophobicity upon alkylation. It should be kept in mind that the sensitivity of the LC/MS analysis is not only determined by the concentration of the peptide in the digest, but is also highly dependent on coeluting contaminants and the ionization potential of the peptide. Therefore, specific amino acids in hemoglobin cannot be ruled out as alkylation sites on the basis of a negative result in these analyses. The analysis of hydrophilic tryptic fragments, which contain residues prone to alkylation by sulfur mustard but which were unfortunately not retained on the column, might be improved by selecting a polymeric reverse phase packing which allows chromatography at a wide pH range. In addition, the use of dibutylamine at pH 5-7 might lead to a considerable increase in retention time, although this will probably also result in a loss in sensitivity. Using CID tandem MS analysis, R-Val-1, R-His-20, β-Val-1, β-His-77, and β-His-97 were identified as alkylation sites. Unfortunately, the site of alkylation could not be established for alkylated R-T6, R-T9, β-T10, and β-T10-S-S-β-T12. For the latter peptide reduction with dithiothreitol prior to analysis did not lead to any improvement. In these peptides histidine could be the site of alkylation. In tryptic fragment β-T10, the Cys-93 residue might have been alkylated. Alkylation of Nterminal valine residues in hemoglobin has been observed for numerous other electrophilic agents (7) and was also established by us for sulfur mustard (8). It should be noted that although the terminal valine was identified as the alkylation site in β-T1, it cannot be ruled out that the histidine residue in this peptide is also alkylated since the two alkylated peptides will probably not be separated in the chromatographic system, resulting in a tandem 2 Noort, D., Hulst, A. G., De Jong, L. P. A., and Benschop, H. P. Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulfur mustard and hemoglobin in vitro, to be published. 3 To the best of our knowledge, antibodies against adducts of nitrogen mustards have not been published. However, our antibodies against N7-[2-[(hydroxyethyl)thio]ethyl]-2′-deoxyguanosine (see ref 5) recognize DNA adducts with the nitrogen mustard bis(2-chloroethyl)methylamine (HN2) only ca. 6-fold less efficiently than sulfur mustard adducts (Dr. G. P. van der Schans, TNO Prins Maurits Laboratory, Rijswijk, The Netherlands, personal communication). Analogously, it cannot be excluded that antibodies against sulfur mustard - amino acid adducts also recognize the corresponding nitrogen mustard adducts.
Tandem MS of Hemoglobin-Sulfur Mustard Adducts
MS spectrum of a mixture. Alkylation of the R-His-20 residue was also found by the group of Burlingame (10) upon treatment of human hemoglobin with styrene 7,8oxide, indicating the accessibility of this particular position. The crystal structure of human hemoglobin indicates that the other four identified alkylation sites are also peripherally located residues, as should be expected. Adducts of glutamic acid, aspartic acid, and cysteine were not found, although we established their formation upon exposure of hemoglobin to sulfur mustard in other studies.2 The esters are probably susceptible to hydrolysis by trypsin. In view of the high reactivity of sulfur mustard (27) toward cysteine, it is remarkable that cysteine adducts could not be detected. Although the presence of alkylated β-T10, containing the reactive Cys93 residue (28), could be demonstrated, a satisfactory CID tandem MS spectrum could not be obtained. In this respect, it is important to note that the predominant alkylation sites in hemoglobin following alkylation with styrene oxide were at peripherally located histidine residues (R-His-20 and β-His-143), as opposed to the more nucleophilic β-Cys-93 residue (10). In conclusion, we demonstrated the applicability of the powerful combination of micro-LC and tandem MS in the structural determination of modified sites of hemoglobin after exposure to sulfur mustard. Partial hemoglobin sequences containing the elucidated modified sites will be used as haptens for raising monoclonal antibodies with high specificity for the alkylated sites. Such antibodies might be useful for immunochemical detection of exposure to sulfur mustard (29) or to nitrogen mustard and derivatives thereof.3
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 787
(9) (10)
(11) (12)
(13)
(14)
(15) (16) (17)
(18)
(19)
Acknowledgment. This work was supported in part by Grant DAMD17-92-V-2005 from the U.S. Army Medical Research and Materiel Command, Fort Detrick, Frederick, MD, and in part by the Directorate of Military Medical Services of the Ministry of Defence, The Netherlands. We gratefully acknowledge Prof. Dr. J. van der Greef (TNO Nutrition and Food Research) for stimulating discussions during the start-up of the project. We thank Gerrit W. H. Moes for preparing [35S]sulfur mustard.
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