Structure Elucidation of DNA Interstrand Cross-Link by a Combination

DNA interstrand cross-link (ICL) agents are widely used for cancer treatment.1,2 Among those cross-linking agents, psoralen. 3r9 and mitomycin C (MC) ...
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Anal. Chem. 2003, 75, 6306-6313

Structure Elucidation of DNA Interstrand Cross-Link by a Combination of Nuclease P1 Digestion with Mass Spectrometry Yuesong Wang and Yinsheng Wang*

Department of Chemistry-027, University of California at Riverside, Riverside, California 92521-0403

DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand crosslink. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5′,8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs. DNA interstrand cross-link (ICL) agents are widely used for cancer treatment.1,2 Among those cross-linking agents, psoralen 3-9 and mitomycin C (MC) 10-12 have been extensively studied. Upon 365-nm UV irradiation, psoralen forms a cross-link between the two thymines at the 5′-TpA site on opposite DNA strands via a [2 + 2] photochemical reaction.3 On the other hand, mitomycin C produces a cross-link between the N2 nitrogen atoms of two guanine residues at the 5′-CpG site in opposing DNA strands after its reductive activation.10 * Corresponding author. E-mail: [email protected]. Fax: (909) 7874713. Tel: (909) 787-2700. (1) Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723-2796. (2) Luce, R. A.; Hopkins, P. B. Methods Enzymol. 2001, 340, 396-412. (3) Kanne, D.; Straub, K.; Hearst, J. E.; Rapoport, H. J. Am. Chem. Soc. 1982, 104, 6754-6764. (4) Yeung, A. T.; Jones, B. K.; Chu, C. T. Biochemistry 1988, 27, 3204-3210. (5) Esposito, F.; Brankamp, R. G.; Sinden, R. R. J. Biol. Chem. 1988, 263, 11466-11472. (6) Matsuo, N.; Ross, P. M. Biochemistry 1987, 26, 2001-2009. (7) Kumaresan, K. R.; Ramaswamy, M.; Yeung, A. T. Biochemistry 1992, 31, 6774-6783. (8) Greenberg, R. B.; Alberti, M.; Hearst, J. E.; Chua, M. A.; Saffran, W. A. J. Biol. Chem. 2001, 276, 31551-31560. (9) Eichman, B. F.; Mooers, B. H. M.; Alberti, M.; Hearst, J. E.; Ho, P. S. J. Mol. Biol. 2001, 308, 15-26. (10) Tomasz, M.; Lipman, R.; Chowdary, D.; Pawlak, J.; Verdine, G. L.; Nakanishi, K. Science 1987, 235, 1204-1208. (11) Weidner, M. F.; Sigurdsson, S. T.; Hopkins, P. B. Biochemistry 1990, 29, 9225-9233. (12) Palom, Y.; Kumar, G. S.; Tang, L.-Q.; Paz, M. M.; Musser, S. M.; Rockwell, S.; Tomasz, M. Chem. Res. Toxicol. 2002, 15, 1398-1406.

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Development of new interstrand cross-link drugs and understanding the mechanism of anticancer activity of those drugs require a sensitive method for locating the site and elucidating the structure of the DNA interstrand cross-link. To pinpoint the sites of ICL in duplex DNA, Hopkins and co-workers 13 developed a powerful method where they separate singly end-radiolabeled, singly cross-linked DNA from unreacted and monoalkylated singlestranded DNA by denaturing polyacrylamide gel electrophoresis (PAGE) followed by random cleavage with iron(II)/EDTA. The cleavage products are then separated on a single-nucleotide resolving, denaturing PAGE.13 The method can reveal the sites of cross-link at single-nucleotide resolution because only those cleavages occurring between the radiolabel and the site of crosslink can result in a labeled fragment that is shorter than the starting single-stranded DNA.11,13-16 However, the method does not yield information about the structural nature of the ICL. The introduction of matrix-assisted laser desorption/ionization (MALDI)17,18 and electrospray ionization (ESI) 19 revolutionized the application of mass spectrometry (MS) for the characterization of biomolecules including modified oligodeoxynucleotides (ODNs). Enzymatic digestion followed by MS analysis has been used for the structure characterization of ODNs bearing a number of different modifications.20-28 In this respect, exonuclease digestion combined with MALDI-TOF MS has been used for locating the (13) Weidner, M. F.; Millard, J. T.; Hopkins, P. B. J. Am. Chem. Soc. 1989, 111, 9270-9272. (14) Millard, J. T.; Weidner, M. F.; Raucher, S.; Hopkins, P. B. J. Am. Chem. Soc. 1990, 112, 3637-3641. (15) Hopkins, P. B.; Millard, J. T.; Woo, J.; Weidner, M. F.; Kirchner, J. J.; Sigurdsson, S. T.; Raucher, S. Tetrahedron 1991, 47, 2475-2489. (16) Millard, J. T.; Weidner, M. F.; Kirchner, J. J.; Ribeiro, S.; Hopkins, P. B. Nucleic Acids Res. 1991, 19, 1885-1892. (17) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yohida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (18) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (19) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (20) Zhang, L.-K.; Rempel, D.; Gross, M. L. Anal. Chem. 2001, 73, 3263-3273. (21) Tretyakova, N.; Matter, B.; Ogdie, A.; Wishnok, J. S.; Tannenbaum, S. R. Chem. Res. Toxicol. 2001, 14, 1058-1070. (22) Zhang, L. K.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2000, 11, 854-865. (23) Wu, H.; Chan, C.; Aboleneen, H. Anal. Biochem. 1998, 263, 129-138. (24) Wu, H.; Morgan, R. L.; Aboleneen, H. J. Am. Soc. Mass Spectrom. 1998, 9, 660-667. (25) Wang, Y.; Taylor, J. S.; Gross, M. L. Chem. Res. Toxicol. 1999, 12, 10771082. (26) Wang, Y.; Taylor, J. S.; Gross, M. L. Chem. Res. Toxicol. 2001, 14, 738745. (27) Wang, Y. Chem. Res. Toxicol. 2002, 15, 671-676. (28) Schrader, W.; Linscheid, M. Arch. Toxicol. 1997, 71, 588-595. 10.1021/ac034683n CCC: $25.00

© 2003 American Chemical Society Published on Web 10/10/2003

sites of modifications.20-23 In addition, digestion with an endonuclease, nuclease P1, followed by tandem mass spectrometry (MS/ MS) analysis of the digestion product has been used for determining the structures of modifications in ODNs.24-27 We anticipate that the latter method should also be useful for elucidating the structure of DNA ICL. In this article, we chose two of the most commonly studied ICL agents, 4,5′,8-trimethylpsoralen (TMP) and mitomycin C, as model systems to develop a general method of nuclease P1 digestion followed by MS/MS for the structure elucidation of duplex ODNs containing an ICL. We will demonstrate that structure information of the ICL can be gained from the production spectra of the nuclease P1 digestion products of these ICLcontaining ODNs. EXPERIMENTAL SECTION Reagents. ODNs used in this study were purchased from Integrated DNA Technologies, Inc. (Coralville, IA) and used without further purification. Mitomycin C was obtained from A. G. Scientific Inc. (San Diego, CA). Nuclease P1, TMP, and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Preparation of DNA ICL Products. We prepared the TMP interstrand DNA cross-link following that reported by Yeung and co-workers.4 A 250-nmol self-complementary ODN d(CGCGCTAGCGCG) was dissolved in 450 µL of reaction buffer, which contains 0.2 mM EDTA, 50 mM NaCl, and 5 mM Tris (pH 7.6); the ODN was annealed by heating to 90-95 °C followed by cooling slowly to room temperature over 2 h. The ODN solution was then diluted to 100 mL with the reaction buffer, to which 1-mL saturated TMP solution in ethanol was added. The resulting solution was dispersed in three 10.4-cm-i.d. Petri dishes and irradiated on ice for 40 min using two 15-W Spectroline light tubes emitting at 365 nm (Spectronics Corp., Westbury, NY). The distance between the light tubes and the Petri dishes was ∼3 cm. After the irradiation, the TMP was removed by extraction with two portions of chloroform (3 mL each) and the ODN was recovered from the aqueous layer by ethanol precipitation. The precipitated ODN was resuspended in doubly distilled water and separated by HPLC, and the collected fractions were dried by using a Savant SpeedVac (Savant Instruments Inc., Holbrook, NY). The mitomycin C interstrand cross-link was prepared following the method reported by Tomasz and co-workers with some modifications.29 Briefly, a 50-nmol self-complementary ODN d(ATATACGTATAT) was dissolved in 1.2 mL of the above reaction buffer and the ODN was annealed in a way similar to that described above. The ODN solution was incubated in an icewater bath, and mitomycin C was then added to the solution with a molar ratio of 48/1 (MC/duplex ODN). Subsequently, five portions of 0.4 M freshly prepared, ice-cooled, deareated Na2S2O4 solution in the reaction buffer was added to the ODN/MC mixture at 10-min intervals (The molar ratio between Na2S2O4 and MC was 1.5/1). The solution was under continuous argon bubbling during the whole reaction process, and the reaction was terminated by exposing the mixture to air. The resulting solution was then dried by the Speed-Vac and purified by reversed-phase HPLC (vide infra). (29) Borowy-Borowski, H.; Lipman, R.; Chowdary, D.; Tomasz, M. Biochemistry 1990, 29, 2992-2999.

The selective preparation of the two orientation isomers of MCICL in non-self-complementary ODNs was achieved according to a procedure reported by Kumar et al.30 A mixture of two complementary ODNs and MC (MC/ODN, 48/1, mole/mole) in a solution of 50 mM NaCl and 10 mM phosphate (pH 7.5) was annealed by heating the solution briefly to 55 °C and cooling it to room temperature slowly. The ODN/MC mixture was cooled in an ice-water bath; to the solution was added ice-cooled aliquots of anaerobic Na2S2O4 solution in 50 mM NaCl and 10 mM phosphate (pH 7.5). The resulting solution was incubated in an ice-water bath and stirred under air for 1 h. The monoalkylated ODNs were isolated from the above reaction mixture by HPLC and annealed with their complementary unmodified ODNs in a solution containing 0.2 mM EDTA, 50 mM NaCl, and 5 mM Tris (pH 7.6). The resulting solution was again cooled in an ice-water bath, and the monoalkylated MC adduct was converted to the MCICL by reduction with freshly prepared Na2S2O4 solution under anaerobic condition.30 HPLC Conditions. The HPLC separation was performed on a system composed of a Hitachi L-6200A pump (Hitachi Ltd., Tokyo, Japan) and a SSI 500 variable-wavelength UV detector (Scientific System Inc., State College, PA). A 4.6 × 250 mm YMC ODS-AQ column (5 µm in particle size and 120 Å in pore size, Waters Co., Milford, MA) was used. A solution of 50 mM TEAA (pH 6.8, mobile phase A) and a mixture of 50 mM TEAA and acetonitrile (70/30, v/v, mobile phase B) were used for the separation. A gradient of 5 min 0-20% B, 65 min 20-50% B, and 25 min 50-80% B was employed and the flow rate was 0.8 mL/ min. Nuclease P1 Digestion. Nuclease P1 was dissolved in doubly distilled water at a concentration of 1 unit/µL. For a typical digestion, 1-µL nuclease P1 solution was added to a 10-µL aliquot containing approximately 5-10 µM ICL-containing ODN. No buffer was added except that present in the commercial preparation of the enzyme. The solution was incubated at room temperature for 20 min, and the digestion mixture was subjected directly to mass spectrometric analysis without further purification. Mass Spectrometry. Most ESI-MS and MS/MS experiments were carried out on an LCQ Deca XP ion trap mass spectrometer (ThermoFinnigan, San Jose, CA). An equal-volume solvent mixture of acetonitrile and water was used for electrospray, and a 2-µL aliquot of sample solution (∼5 µM) was injected in each run. The spray voltage was 3.4 kV, and the capillary temperature was maintained at 200 °C. High-resolution MS/MS experiments were done on an IonSpec HiResESI external ion source FTICR mass spectrometer (IonSpec Co., Lake Forest, CA) equipped with a 4.7-T superconducting magnet and an Analytica electrospray ionization source (Branford, CT). Samples were dissolved in CH3CN/H2O (1/1, v/v) at a concentration of 5 µM and infused by a syringe pump at a flow rate of 1 µL/min. Experiments were done in the negative-ion mode, and the source end plate was kept at 3.0 kV. Ions were accumulated in a hexapole for 1000-1500 ms and transported through a quadrupole ion guide to the analyzer cell by standard pulse sequences. For exact mass measurements in MS/MS, the [M - 2H]2- ion of the analyte of interest and the [M - H]- ion of 2′-deoxyadenosine 5′-monophosphate (pdA) or thymidine 5′(30) Kumar, S.; Johnson, W. S.; Tomasz, M. Biochemistry 1993, 32, 1364-1372.

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Figure 1. HPLC traces for the separation of the interstrand crosslink reaction mixtures: (a) d(CGCGCTAGCGCG) with TMP; (b) d(ATATACGTATAT) with MC. The solid diamond ([) and open triangle (4) indicate the unreacted ODN and the duplex ODN bearing an ICL, respectively.

Figure 2. Negative-ion ESI MS of the nuclease P1 digestion products of TMP-ICL in d(CGCGCTAGCGCG) (a) and MC-ICL in d(ATATACGTATAT) (b). The ions of m/z 306 and 346 in (a) are the deprotonated ions of 2′-deoxycytidine 5′-phosphate and 2′-deoxyguanosine 5′-phosphate; the heterodimer of the two was also observed (m/z 653). Similarly, ions of m/z 321 and 330 in (b) are the deprotonated ions of pdT and pdA; dimers were also observed: [pdT + pdT] (m/z 643), [pdT + pdA] (m/z 652), and [pdA + pdA] (m/z 661).

Chart 1 monophosphate (pdT) were first isolated in the ICR cell by ejection of all other species. These two ions were also used as internal standards for calibration. Sustained off-resonance irradiation/ collisionally activated dissociation (SORI-CAD) of the analyte ion was performed for 1000 ms by a rf burst with an amplitude varied from 1.2 to 2.5 V depending on the analytes and at a frequency around 1000 Hz from the cyclotron frequency of the analyte ion. A 200-ms pulse of nitrogen was introduced at the beginning of the irradiation period (the maximum pressure readout on the ion gauge was 3 × 10-6 Torr). Ions were activated as a result of multiple collisions with pulsed nitrogen gas. After a delay of 2 s, the resulting fragment ions were accelerated for detection by a rf sweep excitation waveform (100 V p-p). The image current was amplified, digitized at an acquisition rate of 2 MHz, and Fourier transformed to yield a mass spectrum. RESULTS AND DISCUSSION Preparation of ICL-Containing Duplex ODNs. We isolated the TMP- and MC-ICL-containing ODNs from the reaction mixtures by reversed-phase HPLC. The 77.1-min peak in Figure 1a and the 80.5-min peak in Figure 1b are the interstrand crosslinks of TMP in d(CGCGCTAGCGCG) and MC in d(ATATACGTATAT), respectively. The presence of a TMP- or MC-ICL in these two duplex ODNs was confirmed by molecular weight measurement using ESI-MS (data not shown). Identities of Nuclease P1 Digestion Products of Duplex ODNs with TMP and MC-ICLs. Previous studies demonstrated that dimeric DNA photoproducts,25,26 thymine glycol,27 and 6308

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monoalkylated MC adduct 31 can block the hydrolysis of the phosphodiester bond that is 3′ to the modified nucleobase. Now we want to investigate whether a MC- or TMP-ICL (Chart 1) also prevents the digestion of the phosphodiester bond 3′ to the crosslinked nucleobases. To this end, we digested the TMP- and MCcross-linked ODNs with nuclease P1, and our results showed that the cross-link indeed blocks the digestion of the phosphodiester bonds that are 3′ to the cross-linked nucleobases. Negative-ion ESI-MS of the digestion products of TMP-cross-linked duplex ODN d(CGCGCTAGCGCG) showed ions of m/z 748 and 499 (Figure 2a), which are the [M - 2H]2- and [M - 3H]3- ions of a tetranucleotide consisting of two dinucleotides d(pTpA) with the two thymines being bridged with the TMP moiety (Chart 2). Similarly, nuclease P1 digestion of a self-complementary duplex ODN d(ATATACGTATAT) containing a MC-ICL gives a tetra(31) Kumar, S.; Lipman, R.; Tomasz, M. Biochemistry 1992, 31, 1399-1407.

Chart 2

Chart 3

Figure 3. Product-ion spectrum of the electrospray-produced [M - 2H]2- ion of the MC-ICL-containing tetranucleotide resulting from nuclease P1 digestion (a) and MS3 of the fragment ions of m/z 1042 (b) and 962 (c) observed in (a).

nucleotide, which is composed of two dinucleotides d(pGpT) with the two guanines being covalently bonded with MC (Structure for the tetranucleotide is shown in Chart 3, and negative-ion ESIMS for the digestion mixture is shown in Figure 2b.). The formation of a tetranucleotide from the nuclease P1 digestion of ICL-containing duplex ODNs can be rationalized from the mechanism of nuclease P1 digestion based on the X-ray cocrystal structure of the enzyme and its substrate complex.32,33 The hydrolysis of the phosphodiester bond is facilitated by nucleophilic attack via a Zn2+-activated water molecule. For the nucleophilic attack to occur, the nucleobase 5′ to the phosphodiester bond to be hydrolyzed must fit into the binding pocket at the active site of the enzyme, where the nucleobase forms π-stacking interaction with a phenylalanine residue.33 The formation of the MC- or TMP-bearing tetranucleotide indicates that the bulky cross-linked nucleobases, i.e., guanine in MC-ICL and thymine in TMP-ICL, cannot be fit into the binding pocket of the enzyme. Therefore, the phosphodiester bonds 3′ to the two crosslinked nucleobases are not hydrolyzed and the digestion yields two covalently bound dinucleotides (Charts 2 and 3). The results are consistent with previous studies with ODNs containing biotin label,24 dimeric DNA photoproducts,25,26 thymine glycols,27 and monoalkylated MC adduct.31 The identity of the nuclease P1-generated covalently bound dinucleotides [i.e., d(pTpA) for the TMP cross-link and d(pGpT) (32) Volbeda, A.; Lahm, A.; Sakiyama, F.; Suck, D. Embo J. 1991, 10, 16071618. (33) Romier, C.; Dominguez, R.; Lahm, A.; Dahl, O.; Suck, D. Proteins 1998, 32, 414-424.

for the MC cross-link] provides some information about the site of the cross-link: it narrows the sites to d(pTpA) and d(pGpT) dinucleotides for the TMP and MC cross-links in the two ODNs being investigated, respectively. ESI-MS/MS of Nuclease P1-Generated Tetranucleotides Containing ICL of MC and TMP. (a) MC-ICL. Next we examined whether the product-ion spectra of the enzymatic tetranucleotides provide structure information of the cross-link. Toward this end, we acquired the product-ion spectrum (Figure 3a) of the ESI-produced [M - 2H]2- ion of the tetranucleotide containing the MC-ICL, and the results show that the glycosidic bond of the modified 2′-deoxyguanosine moiety can cleave to give a pair of ions of m/z 1042 and 499, with the former carrying the cross-linked nucleobase moiety (Chart 3, sites of deprotonation were not shown). The ion of m/z 1042 can further eliminate a neutral HPO3 molecule to give an ion of m/z 962. In addition, fragment ions of m/z 762 and 722 are also produced, which are attributed to the losses of H2O and [HPO3 + H2O], respectively. Moreover, we observed that a pdT can be eliminated as a deprotonated ion to give ions of m/z 321 and 1220 (Figure 3a). The formation of this pair of fragment ions shows that thymine is not cross-linked with the MC. The assignments of fragment ions of m/z 1042, 962, and 722 are supported by high-resolution MS/ MS acquired on an FTICR (Table 1). Other fragment ions observed in Figure 3a were not produced in sufficient abundance under the SORI-CAD conditions on the FTICR. Therefore, the high-resolution measurements for those ions were not attempted. Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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Table 1. High-Resolution ESI-MS/MS of the Tetranucleotide Resulting from the Nuclease P1 Digestion of Self-Complementary Duplex ODN d(ATATACGTATAT) Containing a MC-ICL calc mass

meas mass

dev (ppm)

1042.2358

1042.2415 1042.2417

5.5 5.7

962, [dGpdT + Gua + MC]-

962.2695

962.2702 962.2701

0.7 0.6

722, [M - HPO3 - H2O]2-

721.6554

721.6578 721.6578

3.3 3.3

1042, [pdGpdT + Gua + MC]-

MS/MS/MS (MS3) of fragment ions of m/z 1042 and 962 lends further support for the assignments of the two ions. In the MS3 of the former (Figure 3b), we observed neutral losses of H2O, HPO3, [HPO3 + H2O], and thymine, which yield ions of m/z 1024, 962, 944, and 916, respectively. Furthermore, the cross-linked nucleobase moiety can be lost either as a neutral molecule or as a deprotonated ion, which give ions of m/z 499 and 542, respectively. In the MS3 of the ion of m/z 962 (Figure 3c), we observed two very abundant fragment ions of m/z 836 and 542, which are attributed to the loss of a neutral thymine and the formation of the deprotonated ion of the cross-linked nucleobase moiety, respectively. The elimination of the cross-linked nucleobase portion from the ions of m/z 1042 and 962 demonstrates that the glycosidic bond of one of the two 2′-deoxyguanosines was ruptured upon the production of these two ions. The elimination also demonstrates that the two guanines are crosslinked with MC. (b) TMP-ICL. We also characterized the TMP-ICL-bearing tetranucleotide by ESI-MS/MS. The most abundant ion in the product-ion spectrum of the ESI-produced [M - 2H]2- ion of the tetranucleotide containing TMP-ICL (Figure 4a) is due to the loss of a pdA (m/z 1166), whose complementary ion of m/z 330 is also produced abundantly. This is in accordance with the fact that adenine is not involved in the cross-link. The ion of m/z 1166 can eliminate an adenine to yield an ion of m/z 1031, which can further lose [HPO3 + H2O] to give a product ion of m/z 933. Similar to the fragmentation of the tetranucleotide containing a MC-ICL, we observed the cleavage of the glycosidic bond of the modified thymidine, which yields a pair of complementary ions of m/z 988 and 508. The cleavages for the formation of major fragment ions are shown in Chart 2, and the assignments of most fragment ions are again supported by exact mass measurements (Table 2). MS3 experiment furnishes further evidence for the identity of the ion of m/z 988 and yields information about the structure nature of the ICL (Figure 4b). We found that this ion can lose a pdA moiety to generate an ion of m/z 657. This elimination again shows that pdA is not involved in the cross-link. In addition, we observed neutral losses of thymine and adenine, which give ions of m/z 862 and 853, respectively. The ion of m/z 862 is also present in Figure 4a, and it can further eliminate the TMP moiety to yield an ion of m/z 634. The formation of ions of m/z 862 and 634 shows that collisional activation in the ion trap can revert the [2 + 2] photochemical reaction. The sequential losses of thymine and TMP demonstrate that the glycosidic bond of one of the two 6310 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

Figure 4. Product-ion spectrum of the electrospray-produced [M - 2H]2- ion of the TMP-ICL-containing tetranucleotide resulting from nuclease P1 digestion (a) and MS3 of the fragment ion of m/z 988 (b) observed in (a). Table 2. High-Resolution ESI-MS/MS of the Tetranucleotide Resulting from the Nuclease P1 Digestion of Self-Complementary Duplex ODN D(CGCGCTAGCGCG) Containing a TMP-ICL calc mass

meas mass

dev (ppm)

1166, [M - pdA-]-

1166.2310

1031, [M - Ade - pdA-]-

1031.1766

933, [M - Ade - HPO3 H2O - pdA-]862, [M - pdTpdA]-

933.1997

632, [M - Ade - HPO3 - H2O]2-

631.6300

582.6, [M - pdA]2-

582.6116

508, [M - pdTpdA - TMP]-

508.0635

1166.2447 1166.2434 1031.1829 1031.1815 933.2061 933.2050 862.1911 862.1900 631.6316 631.6313 582.6136 582.6135 508.0653 508.0646

11.7 10.6 6.1 4.8 6.9 5.7 7.1 5.8 2.5 2.1 3.4 3.3 3.5 2.2

ions, assignments

862.1850

thymidines has been cleaved upon the formation of the ion of m/z 988, which supports our assignment of this fragment ion. Distinction of two Orientation Isomers of MC-ICL. Because the two moieties of MC (1′′ side and 10′′ side, Chart 1) that form a cross-link with the amino group of guanines are not the same, two orientation isomers of ICL, i.e., with guanine in one strand being cross-linked to the 1′′ or 10′′ side of MC, can be generated if the two opposing DNA strands are not self-complementary.30 In this respect, Tomasz and co-workers 30 developed a method for the selective preparations of individual isomers and demonstrated that the two orientation isomers have different susceptibilities toward cleavage by AluI restriction endonuclease. We reason that the tetranucleotides emanating from nuclease P1 digestion of ODNs containing the two isomeric MC-ICLs will not be the same if the two nucleobases that are 3′ to the crosslinked guanines are not identical (Figure 5). Given that production spectra of the enzymatic tetranucleotides provide structure

Figure 5. Preparation of two orientation isomers of MC-ICL in d(AAAAGCGATATA)/d(TATATCGCTTTT) and the nuclease P1 digestion products of the two isomer-bearing duplex ODNs.

information of the ICLs, it is important to determine whether we can extend the MS/MS method to the distinction of the two orientation isomers of the MC-ICL. To this end, we adopted a procedure reported by Kumar et al. 30 and prepared the MC-ICLs by using three duplex ODNs that are not self-complementary, i.e., d(TATATCGXTTTT)/d(AAAAYCGATATA), where X:Y is a G:C, C:G, or T:A base pair. The selective preparations of individual isomers are based on mitomycin C forming a monoalkylated adduct with guanine specifically through its 1′′ carbon atom while the duplex ODN/ MC mixture is treated with Na2S2O4 under aerobic conditions (Figure 5).31 The monoalkylated MC-containing single-stranded ODNs were isolated from the reaction mixture by HPLC, annealed with their complementary strands, and treated with Na2S2O4 under anaerobic conditions (details shown in the Experimental Section). The resulting MC-ICL-containing duplex ODNs were again isolated from the latter reaction mixtures by reversed-phase HPLC. The HPLC traces for the separation of the mixture for the monoalkylation reaction and those for the conversions of the monoalkylated products to ICLs for duplex d(TATATCGCTTTT)/ d(AAAAGCGATATA) are shown in Figure 6. It is worth noting that the monoalkylated d(TATATCGCTTTT) cannot be completely resolved from its unmodified precursor by HPLC (Figure 6a). The mixture of the unmodified and monoalkylated d(TATATCGCTTTT) was used directly for converting the monoalkylated adduct to the ICL, and it turned out that the presence of the unmodified

Figure 6. HPLC traces for the separation of (a) the monoalkylation reaction mixture of MC with d(AAAAGCGATATA)/d(TATATCGCTTTT), (b) the reaction mixture for the conversion of monoalkylated adduct of d(AAAAGCGATATA) to ICL, and (c) the reaction mixture for the conversion of monoalkylated adduct of d(TATATCGCTTTT) to ICL. The solid diamond ([) and open triangle (4) in (b) and (c) denote the unreacted single-stranded ODN and the duplex ODN bearing an ICL, respectively.

d(TATATCGCTTTT) did not affect the formation and isolation of the desired ICL (Figure 6c). Nuclease P1 digestion of the duplex ODN containing one or the other isomer of the MC-ICL again gives a tetranucleotide bearing the cross-linked nucleobase moiety as demonstrated by ESI-MS analysis (data not shown). The product-ion spectra of the [M - 2H]2- ions of the tetranucleotides are similar to that of the [M - 2H]2- ion of the tetranucleotide originated from the MCICL-containing self-complementary duplex ODN (Figure 7). Now we want to focus our discussion on three pairs of fragment ions (i.e., ions of m/z 651/663, 947/971, and 1027/1051) because their relative abundances are different for the two orientation isomers. Among them, doubly charged product ions of m/z 651 and 663 are attributed to the losses of [Ade + H2O + HPO3] and [Cyt + H2O + HPO3] (Ade and Cyt are adenine and cytosine), respectively. The ions of m/z 1027 and 1051 are due to cleavages of glycosidic bonds of the two modified 2′-deoxyguanosines (vide supra, cleavages shown in insets of Figure 7), which can further eliminate a familiar HPO3 molecule to yield ions of m/z 947 and 971, respectively. The assignments of all these fragments are supported by high-resolution ESI-MS/MS acquired on the FTICR (results for the product-ion spectrum of one tetranucleotide is shown in Table S1 in the Supporting Information). Our results show that the relative propensities for the cleavages of the two glycosidic bonds of the modified 2′-deoxyguanosines (i.e., the formation of ions of m/z 1027 and 1051) are different for Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

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Figure 7. Product-ion spectra of the electrospray-produced [M - 2H]2- ions of the tetranucleotide resulting from the nuclease P1 digestion of d(TATATCGCTTTT)/d(AAAAGCGATATA) containing the two orientation isomers of MC-ICL (Structures for the two orientation isomers are shown in the insets.)

the two orientation isomers. Particularly, we observed that the cleavage of the glycosidic bond that is close to the 1′′ side of the MC occurs more readily than the glycosidic bond that is close to the 10′′ side of the MC. Fragment ions originating from further elimination of the HPO3 moiety (ions of m/z 947 and 971) follow a similar trend (Figure 7). We also characterized the tetranucleotides from the other two MC-ICL-bearing non-self-complementary duplex ODNs by MS/MS, and the results are similar as those discussed above (data shown in the Supporting Information). In addition to the different susceptibility of glycosidic bond cleavage, the [M - 2H]2- ions of the enzymatic tetranucleotides of the two orientation isomers show different relative abundances of two fragment ions of m/z 651 and 663 (Figure 7). The formation of these ions is dependent on the identities of the unmodified nucleobases: The elimination of [Gua + H2O + HPO3] or [Ade + H2O + HPO3] is more facile than that of [Cyt + H2O + HPO3] or [Thy + H2O + HPO3] (Gua and Thy are guanine and thymine). Beyond that, we also observe that the elimination is more facile while unmodified nucleobase is in close proximity to the 10′′ side than while it is close to the 1′′ side of the MC moiety (Figure 7 and the data shown in the Supporting Information). The reason why this elimination and the glycosidic bond cleavage have different preferences is not clear. Because the distinction of the two isomers lies in differences in relative abundances of fragment ions, we need to determine whether the product-ion spectra of the two isomers are reproducible. To this end, we acquired the product-ion spectra at five different normalized collisional energies and it turned out that the 6312 Analytical Chemistry, Vol. 75, No. 22, November 15, 2003

Figure 8. Ratios of relative abundances of product ions observed in the MS/MS of the [M - 2H]2- ions of tetranucleotides containing the two orientation isomers of MC-ICL as a function of normalized collisional energies. Error bars denote the standard deviations calculated from three independent measurements. The solid and open squares designate that the tetranucleotides were from the digestion of the MC-ICL with the d(AAAAGCGATATA) and d(TATATCGCTTTT) being covalently bonded to the 1′′ side of the MC, respectively.

differences remain at these collisional energies (Figure 8). The only exception is that, at 20% normalized collisional energy, the relative ion intensity ratios of I947/(I947 + I971) for the two isomers are reversed compared to other collisional energies (Figure 8b). This observation is also true for the ion intensity ratios for the corresponding ions in the MS/MS of the [M - 2H]2- ions of the two isomeric tetranucleotides in other sequence contexts (Figures S3 and S6 in the Supporting Information). It is worth mentioning that the differences in relative abundances of fragment ions resulting from glycosidic bond cleavages and those from the subsequent loss of HPO3 for the two orientation isomers are relatively small considering the magnitudes of the standard deviations for the measurement. The differences for the relative abundances of the fragment ions resulting from the elimination of [nucleobase + H2O + HPO3] for the two orientation isomers, however, are significant. Therefore, we conclude that the fragment ions emanating from the elimination of [nucleobase + HPO3 +

H2O] are the analytically most useful for the distinction of the two orientation isomers. Here we differentiate the two isomers by using two different nucleobases that are 3′ to the cross-linked guanine as mass tags. The method is useful only when an agent can form 3′-3′ crosslink (i.e., MC), and the two nucleobases 3′ adjacent to the crosslinked nucleobases are not identical. The method, however, will be generally useful if isotope-labeled nucleosides are used.34 CONCLUSIONS We showed that nuclease P1 digestion of duplex ODNs containing an ICL gives a tetranucleotide bearing the cross-linked nucleobases, which can be rationalized from the X-ray cocrystal structure of nuclease P1 and its substrate ODN. This result is also consistent with previous studies of ODNs containing a biotin label,24 dimeric DNA photoproducts,25,26 and thymine glycols.27 We demonstrated that the resulting tetranucleotides are amenable to ESI-MS/MS characterization, which provides structure information of the ICL. In this respect, we showed that the product-ion spectra of the deprotonated ions of the enzymatic tetranucleotides are distinctive for the two orientation isomers of MC-ICL. The coupled enzymatic digestion/tandem MS approach described in this paper is complementary to the method developed (34) Wang, Y.; Gross, M. L.; Taylor, J. S. Biochemistry 2001, 40, 11785-11793.

by Hopkins et al. 13 toward the characterization of DNA ICL. The Hopkins method facilitates unambiguous determination of the sites of the ICL; our method, however, only narrows the sites to a dinucleotide sequence context. In addition, the method developed by Hopkins and co-workers 13 does not offer information about the structure nature of the cross-link; the coupled nuclease P1 digestion/mass spectrometry method described here, however, provides such information by giving the molecular weight and the fragmentation behavior of the enzymatic tetranucleotide. The enzymatic digestion is done without supplying additional buffer, and the sample can be analyzed directly without HPLC purification or cleanup. The method, therefore, is very efficient. ACKNOWLEDGMENT We thank the National Institutes of Health for supporting this research (R01 CA 96909). SUPPORTING INFORMATION AVAILABLE The preparation and MS/MS characterization of the two orientation isomers of MC-ICL in other two duplex ODN sequences. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 23, 2003. Accepted September 5, 2003. AC034683N

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