Anal. Chem. 1994,66,1274-1285
Structural Characterization of Polycyclic Aromatic Hydrocarbon Dihydrodiol Epoxide DNA Adducts Using Matrix-Assisted Laser Desorption/ Ionization Fourier Transform Mass Spectrometry E. A. Stemmier' Department of Chemistry, Bowdoin CollegeJBrunswick, Maine 040 1 1
M. V. Buchanan, G. B. Hurst, and R. L. Hettich' Oak Ridge National Laboratory, P.O. Box 2008, Oak RMge, Tennessee 37831-6120 Matrix-assisted laser desorption/ionization(MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied for the structural characterization of four polycyclic aromatic hydrocarbon dihydrodiol epoxide (PAHDE) adducts, including the 5,ddimethylchrysene DE adduct of 2'-deoxyadenosine, the 5-methyl- and 5,ddmethylchrysene DE adducts of 2'-deoxyguanosine, and the benzo[a]pyrene-DE adduct of 2'-deoxyguanosyl 3'-phosphate. Measurement of positive and negative ion mass spectra, accurate mass determinations, and CID experiments were carried out using 10-40 ng (20-70 pmol) of sample. An evaluation of five MALDI matrices showed that matrix selection can be used to control the degree of analyte fragmentation. Three MALDI matrices commonly used for the analysis of proteins (sinapinic acid, ferulic acid, 2,5-dihydroxybenzoic acid) gave positive ion adduct mass spectra showing protonated or sodiated molecular ions accompanied by abundant, structurally informative fragment ions. Fragmentation was significantly reduced when working with two matrices used for oligonucleotide analysis (an anthranilic-nicotinic acid mixture and 3-hydroxypicolinic acid). Using the CID capabilities of FTMS, isolation and activation of the MALDI-produced ions was used to provide additional structural information. While characteristic negative ions were not detected for the adenosyladduct, the guanosyl and guanosyl3'-phosphate adducts gave [M - HI- ions when the anthranilic-nicotinic acid matrix mixture was used. The guanosyl adducts also showed [M - H - 2H2Or fragments. Compared with FAB or FAB-MS/MS for the analysis of underivatized PAH-DE adducts, MALDI-FTMS signals are long-lived, the direct MALDI-FT mass spectra show more structurally informativefragments, and accurate mass and CID experiments require lower sample quantities. The structural characterization of biomolecules modified by interactions with xenobiotics or radiation is essential to understanding the biochemical basis of cancer. The identification of carcinogen-DNA adducts provides information relevant to the identification of carcinogenic agents and the mechanisms by which carcinogens are activated.' Polycyclic aromatic hydrocarbons (PAH) are one class of chemical (1) Chemical Carcinogens; Scarle, C. E., Ed.; ACS Monograph 173; American Chemical Society: Washington, DC, 1976.
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carcinogens that has been studied intensely because of their widespread presence in the environment and the high level of carcinogenic or mutagenic activity exhibited by particular isomers.24 Studies indicate that PAH are activated by two major mechanisms involving either oxidation to dihydrodiol epoxide (DE) intermediates5or one-electron oxidation to form intermediate radical cations.6 The former mechanism results in the production of trihydroxy tetrahydro PAH adducts, where covalent attachment of the PAH-DE occurs at the exocyclic amino group of the DNA base. In the elucidation of mechanisms of carcinogen activation and reactions with DNA, much work has been directed at the identification of adducts produced by in vitro reactions using HPLC separation techniques coupled with NMR, circular dichroism (CD), UV/visible, and mass spectrometric characterizati~n.~.~ Because of the limited quantities of material that can be obtained from in vivo experiments, coupled with the highly polar nature of adducted nucleic acid components, the development of methods for determining in vivo DNA modifications has provided significant challenges to researchers.'~~Methods with sufficient sensitivity to detect low levels of adducts (32P-postlabeling, immunoassays, fluorescence assay^^.^) provide little or no structural information for unknown adducts; thus, highly sensitive, structurally informative analytical techniques are needed for the general characterization of DNA modifications. Mass spectrometric approaches offer structural specificity; however, the analysis of DNA adducts by MS has been limited by the highly polar, thermally labile nature of nucleoside and nucleotide a d d u c t ~ . ~ -In l ~early work, these problems were (2) Dipple, A. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R. G., Ed.; ACS Symposium Series 283; American Chemical Society: Washington, DC, 1985; pp 1-17. (3) Harvey, R. G.; Geacintov, N. E. Ace. Chem. Res. 1988, 21, 66-13. (4) Harvey, R. G.Ace. Chem. Res. 1981, 14, 218-226. (5) Gcacintov, N. E. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R. G., Ed.; ACS S y m p i u m Series 283; American Chemical Society: Washington, DC, 1985; pp 107-124. (6) Cavalieri, E.; Rogan, E. Enuiron. Health Perspect. 1985, 64, 68-84. (7) Jeffrey, A. M. In Polycyclic Hydrocarbons and Carcinogenesis;Harvey, R. G.,Ed.;ACSSymposiumScries283;AmericanChemicalSociety: Washington, DC, 1985; pp 187-208. (8) Cadet, J.; Weinfeld, M. Anal. Chem. 1993, 65, 675-682A. (9) Chiarelli, M. P.; Lay, J. O., Jr. Mass Speczrom. Reo. 1992, 11, 447493. (IO) McCloskey, J. A.; Crain, P. F. Int. J. Mass Spectrom. Ion Processes 1992, 1181119, 593-615. (1 1) Crain, P. F. Mass Spectrom. Reu. 1990, 9, 505-554.
0003-2700/94/03661274$04.50/0
0 1994 American Chemical Society
addressed by the use of chemical derivatization, such as permethylation or per~ilylation,~J~J~ or field desorption (FD)/ ionization t e c h n i q ~ e s . ~These J ~ two approaches were used for the characterization of adducts produced by reactions of benzo[a]pyrene (BaP) DE withcalfthymus DNA.12-14 While FD has been supplanted by other desorption/ionization techniques, derivatization continues to be applied to the trace level detection of targeted DNA or protein adducts,8J5 which are often tagged with electrophoric groups for sensitiveelectron capture negative ion detection.I5J6 For example, BaP-DE adducts have been detected following acid hydrolysis of the tetrahydrotetrol from DNA followed by oxidation and derivatization with pentafluorobenzyl bromide.l6 While derivatization may be useful for the quantitative determination of targeted adducts, the analysis of underivatized materials is preferred for the characterization of adducts of unknown structure because of structural alterations that may result from the derivatization process. Fast atom bombardment (FAB)I7J8has provided a means of ionizing nonderivatized adducted nucleosides and nu~leotides.~-~'J9-~' FAB mass spectra of underivatized adducts show protonated molecular ions, MH+, accompanied by one fragment, the base (aglycon) ion, produced by cleavage at the glycosidic bond. In general, the spectra are devoid of other structurally informative fragment ions. The two major limitations of FAB mass spectra, namely, reduced structural information and interferences from the FAB matrix, may be circumvented by the use of collisionally induced dissociations (CID or MS/MS).9J0g20.21In structure confirmation studies, FAB has been used for the analysis of reference adducts formed by the reaction of BaP-DE with deoxyribonucleosides.22In studies where sensitivity has been addressed, sample requirements for the measurement of full-scan FAB spectra of amino PAH adducts have been reported at the 100-ng (0.2 nmol) level,20with 300-500 ng (0.7-1 1 nmol) of material recommended for the analysis of unknowns.21 Chemical derivatization has been required to reduce FAB sample requirements to the low-nanogram range.20.21For the analysis of underivatized adducts, improvements in sensitivity have been achieved using continuous-flow (CF) FAB23 and capillary zone electrophoresis (CZE) with CF-FAB,z4 where multiple reaction monitoring for the constant neutral loss of deoxyriboseresulted in the detection of low-picogramquantities of target add~cts.2~ Matrix-assisted laser desorption/ionization (MALDI) mass s p e ~ t r o m e t r y , ~introduced ~-~~ by Karas and Hillenkamp in (12) Straub, K. M.; Burlingame, A. L. Eiomed. MassSpectrom. 1981,8,431-435. (13) Straub, K. M.; Meehan, T.; Burlingame, A. L.; Calvin, M. Proc. Narl. Acad. S C ~U.S.A. . 1911, 74, 5285-5289. (14) Straub,K. M.; Bur1ingame.A. L. Adu.MassSpectrom. 1980,8E,1127-1134. (15) Wishnok, J. S.Anal. Chem. 1992,64, 1126-1135A. (16) Allam, K.; Abdel-Baky, S.;Giese, R. W. AMI. Chem. 1993,65,1723-1727. (17) Barber, M.; Bordoli, R. S.;Sedgwick, R. D.; Tyler, A. N . J. Chem. Soc., Chem. Commun. 1981,325-326. (18) Fenselau, C.; Cotter, R. J. Chem. Rev. 1981,87, 501-512. (19) Mitchum. R. K.;Evans, F. E.; Freeman, J.P.;Roach,D.Int.J.MassSpectrom. Ion Processes 1983, 46, 383-386. (20) Bryant M. S.;Lay, J. O., Jr.; Chiarelli, M. P. J . Am. SOC.Mass Spectrom. 1992.3, 360-371. (21) Annan, R. S.; Giese, R. W.; Vouros, P. AMI. Eiochem. 1990, 191, 86-95. (22) RamaKrishna,N. V. S.;Gao, F.; Padmavathi,N. S.;Cavalieri, E. L.; Rogan, E. G.; Cerny, R. L.; Gross, M. L. Chem. Res. Toxicol. 1992,5, 293-302. (23) Wolf,S. M.;Annan, R.S.;Vouros,P.;Giese,R.W. BiologicalMassSpectrom. 1992, 21, 647-654. (24) Wolf, S.M.; Vouros, P.; Norwood, C.; Jackim, E. J . Am. SOC.MassSpectrom. 1992, 3,757-761. (25) Karas, M.; Hillenkamp, F. Anal. Chem. 1988.60, 2299-2301.
1987,28 has rapidly become a valuable technique for the analysis of high molecular weight biomolecules, with sensitivities in the low-femtomole range reported for the analysis of proteins.29 Because of this sensitivity, MALDI has the potential to become a valuable method for the determination of altered nucleic acid components. As an indication of the sensitivity of this technique, the time-of-flight (TOF) mass spectrum of an arylamine adduct of deoxyguanosine,measured using 20 fmol loaded on the probe, has recently appeared in the l i t e r a t ~ r e .This ~ sensitivity is more compatible with the quantities of adducted materials that may be obtained from in vivo experiments. MALDI is typically implemented using a TOF mass analyzer; however, the low mass resolution and the inability to conduct MS/MS experiments on typical TOF systems will limit the structural information that may be obtained for low molecular weight DNA adducts. In contrast, Fourier transform ion cyclotron resonance mass spectrometry (FTMS)30-32with MALDI can provide detailed structural information for biomolecules using the highresolution, accurate mass, and ion-trapping capabilities of FTMS.33 Past studies from our laboratory have shown that MALDI-FTMS, with 266- or 355-nm radiation from a pulsed Nd:YAG laser, can be used for the structural characterization of normal and modified nucleic acid In addition to high-resolutionand CID experiments,ion-molecule reactions may be exploited to enhance structural information. For example, hydrogen4euterium (H-D) exchange reactions have been used to provide information regarding methyl substitution sites for modified g ~ a n i n e s . ~ ~ In this report, the application of MALDI-FTMS at 355 nm to the structural characterization of PAH-DE nucleoside and nucleotide adducts is described. First, experimental and mass spectral considerations that influence selection of an appropriate MALDI matrix are addressed, including a discussion of matrix effects on adduct fragmentation. Second, the fragment ions detected in the MALDI-FT mass spectra of group of PAH-DE nucleoside and nucleotide adducts are discussed in detail using information provided by accurate mass measurements and CID experiments.
EXPER IMENTAL SECTION Instrumentation. An Extrel FTMS-2000 system (Millipore-Extrel FTMS, Madison, WI) equipped with a 3-T superconducting magnet and a differentially pumped dual(26) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. AMI. Chem. 1991,63, 1193-1203A. (27) Karas, M.; Bahr, U.;Gimmann, U.MassSpectrom. Reu. 1991,10,335-357. (28) Karas, M.; Bachmann, D.; Bahr, U.;Hillenkamp, F. In?. J. Mass Spectrom. Ion Processes a981, 78, 53-68. (29) Karas, M.; Ingendoh, A.; Bahr, U.;Hillenkamp, F. Eiomed. Mass Spectrom. 1989, 18, 841-843. (30) Marshall, A. G.; Grosshans, P. B. AMI. Chem. 1991,63, 215-229A. (31) Wilkins, C. L.; Chowdhur, A. K.; Nuwaysir, L. M.; Coatcs, M. L. Mass Spectrom. Rev. 1989, 8, 67-92. (32) Buchanan, M. V.; Comisarow, M. B. In Fourier Transform Mass Spectrometry: Evolution, Innwation, and Applications; Buchanan, M. V., Ed.; ACS Symposium Series 359; American Chemical Society: Washington, DC, 1985; pp 1-20. (33) Buchanan, M. V.; Hettich, R. L. AMI. Chcm. 1993,65, 245-259A. (34) Hettich, R. L.; Buchanan, M. V. Inr. J. Mass Spectrom. Zon Processes 1991, 11I , 365-380. (35) Hettich, R.; Buchanan, M. J. Am. Soc. Mass Spectrom. 1991, 2, 402-412. (36) Stemmlcr, E. A.; Hettich, R. L.; Hunt, G. B.; Buchanan, M. V. RapidCommun. Mass Spectrom. 1993, 7 , 828-836. (37) Nourse, B. D.; Hettich, R. L.; Buchanan, M. V. J . Am. Soc. Mass Spectrom. 1993, 4, 296-305.
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ion cell was used for MALDI experiments. A Spectra Physics DCR-11 pulsed Nd:YAG laser (Mountain View, CA) was operated in the Q-switched mode to generate frequency-tripled radiation at 355 nm. Samples were applied to the outer edge of a 1.7-cm-diameter stainless steel removable probe tip. Sample irradiance (106-107 W cm-2 maximum) was adjusted using a variablcattenuator (Model 935-10,Newport, Fountain Valley, CA). The focused laser spot size was approximately 250 X 250 pm, and the laser pulse duration was nominally 8 ns. An ion deceleration technique, first described by Castoro et al.,38was used in this study. This technique has been adapted for our 3-T FTMS system and is described in a recent publication.36 Conditions for ion deceleration involved setting the conductance limit potential (VCL)of the dual-cell FTMS at -9.0 (negative ions) or +9.0 V (positive ions) with the front trapping plate (VS)and probe potentials held at 0 V. The Nd:YAG laser is triggered by the FTMS after the deceleration potentials are established. The conductance limit and front trapping plate potentials are changed to their final trapping values (f3.0 V for the experiments described here) following a variable ion deceleration time delay. The total delay time between the laser trigger and adjustment of VCLand Vs to the final trapping potentials ranged from approximately 250 to 450 pus; however, this delay includes the time between the laser'trigger and laser firing, which ranges from approximately 250 to 350 ps depending upon the Nd:YAG Q-switch delay setting. The delay times ( f D ) used in this study, 50 f 5 ps, have been corrected to reflect only the time between laser firing and ion trapping. Source side detection was used in all experiments following wide-band stored-waveform inverse Fourier transform (SWIFT) e x ~ i t a t i o n .Pressure ~~ in the source cell was typically (1-2) X lo-' Torr, with pressure measurements made using an uncalibrated ionization gauge tube. Typically, 25 single-shot spectra were averaged to improve the signal-to-noise ratio, although complete spectra could be measured with a single laser shot. Accurate mass measurements were made using 2'deoxycytidylyl-(3'-5')-thymidine (d(3'-CT-5'); Sigma Chemical Co., St. Louis, MO) added as an internal calibrant to the matrix-analyte mixture at a 1:1d(3'XT-Y):adduct mole ratio. The [M Na]+, [M + 2Na - HI+, and [M + Na C ~ H ~ I N ~ ions O ~ ]from + d(3'-CT-5') at m / z 554.12586, 576.10781, and 345.04582 were used for mass calibration under medium resolution (4-1 8K fwhm) conditions. Either the protonated cytosine base from d(3'-CT-5') or the protonated guanine or adenine bases from the PAH-DE adduct were used as low-mass calibrant ions. CID experiments were performed by isolating the ion of interest using a SWIFT waveform followed by acceleration (100-500-eV translational energy range) into the argon collision gas at a static pressure of -2 X 10-6 Torr. Adducts and Sample Preparation. 4-(Deoxyadenosin-Myl)-5,6-dimethyl- 1,2,3-trihydroxy- 1,2,3,44etrahydrochrysene (l),4-(deoxyguanosin-N2-yl)-5,6-dimethyl1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene (2), and 4-(deoxyguanosinN2-yl)-5-methyl-l,2,3-trihydroxy-l,2,3,4-tetrahydrochrysene (3) (Figure 1) were obtained from Dr. Steve Hecht
+
(38) Castoro, J. A,; Kbter, C.; Wilkins, C. Rapid Commun. MassSpectrom. 1992, 6, 239-241. (39) Chen, L.; Wang, T.-C. L.; R i m , T.L.; Marshall, A. G. Anal. Chem. 1987, 59, 449-454.
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0 CH,
k"d
MW557
Y HO
MW573
0'
(1) 0
/I
--
Figure 1. Structures of the PAH diol epoxide adducts: qdeoxyadenosin-~-yl)-5,6dlmethyl-l,2,3-trihydroxy-l,2,3,4-tetrahydrochryys8ne (l), e(deo~WSi1+#yl)55,gdimethyC1,2,3-~y&0~y-l,2,3,4( 2 ) , 4 4 ~ ~ ~ ~ ~ 1,2,3,4tetrahydrochrysene (31, and lOR-(Mdeoxyguanosyl 3'phosphete~7R,8S,9Slhydroxy-7,8,9,lD-tetrahydrobenro[a] pyrene (4).
and Dr. Shantu Amin of the American Health Foundation as water-methanol solutions with concentrations in the 20-70 pM range. The isolation and identification of these compounds from reactions of the dihydrodiol epoxide precursors with calf thymus DNA have been described.@'2 The samples were analyzed by HPLC prior to analysis to verify purity. The 1OR-(NZ-deoxyguanosyl3'-phosphate)-7R,8S,9S-trihydroxy7,8,9,10-tetrahydrobenzo[u]pyrene(4) sample was obtained from the National Cancer Institute Chemical Carcinogen Repository (Kansas City, MO) as a 70 pM solution in 0.05 M Tris-HC1 buffer, pH 7.0. All matrix compounds were obtained from Aldrich Chemical Co. (Milwaukee, WI). Samples were prepared for MALDI analysis by mixing the matrix solution (0.5-1 pL) with the analyte solution (1 .O pL) to obtain a matrix to analyte mole ratio in the range of 104:l-103:l. Matrix solution concentrations, 1.5 M for 2 3 dihydroxybenzoic acid (DHBA) and 0.5 M for 3-hydroxypicolinic acid (HPA), 0.09 M for 4-hydroxy-3-methoxycinnamicacid (ferulic acid, FA), and 0.06 M for 3,Sdimethoxy4-hydroxycinnamic acid (sinapinic acid, SA) were close to the solubility limit for each compound in 1:l CHsCN-H20. The anthranilic acid (AA)-nicotinic acid (NA) matrix mixture contained a 4:l mole ratio of the two matrices, with a total concentration of 0.3 M. The analyte-matrix mixture was applied to the probe tip, resulting in surface coverage of -7 f 2 mm* for a 1-pL sample application, and samples were air-dried without assistance. The application of ammoniumactivated ion-exchange beads (Dowex 50W-X12, 100-200 mesh; Bio Rad Laboratories, Richmond, CA) to the probe surface prior to application of the analyte-matrix mixture (40) Misra, E.;Amin, S.;Hecht, S. S.Chem. Res. Toxicol. 1992, 5, 248-254. (41) Misra, E.;Amin, S.; Hecht, S.S.Chem. Res. Toxicol. 1992, 5, 242-241. (42) Melikian. A. A,; Amin, S.; Hecht, S.S.;Hoffmann, D.; Pataki, J.; Harvey, R. G. Cancer Res. 1984,44, 2524-2529.
1
,
2
was used to reduce the degree of ~odiation.4~ Alternatively, a modification of a C1g reversed-phase chromatographic desalting procedure was used.44 Our approach used SPEC VC C-18 15- or 1.5-mg disks (TOXI-LAB, Irvine, CA) that were supported in a 1.5-mL centrifuge tube. The disks were washed with 50 pL of CH30H, 50 pL of CH3CN, and 50 pL of H2O; followed by the addition of 1-2 pg of the nucleoside adduct. After three 50-pL washes with H20, the nucleoside adduct was eluted with three 50-pL CH3CN washes. Quantitative yields for this cleanup procedure were not evaluated; however, reconstituting the sample to the same volume as the original sample resulted in the production of MALDI spectra that were of comparable or slightly lower intensity.
multiply charged49 ion production. Helpful discussions of MALDI matrices have appeared in a few recent publication~.~~~~ For the FTMS analysis of lower molecular weight materials, such as PAH-adducted nucleosides, additional experimental and mass spectral considerations become important. Most MALDI matrices sublime under vacuum, and this process should occur slowly to provide the lowest pressure for highresolution FTMS measurements. To facilitate the acquisition of CID mass spectra, the matrix should produce consistent, long-lasting analyte signals. On our system, which is not equipped with viewing optics, location of the crystalline sample on the probe tip is simplified if the analyte-matrix sample crystallizes uniformly on the probe tip. Regarding the interpretation of MALDI-FT mass spectra of DNA adducts, RESULTS AND DISCUSSION the matrix should ideally produce a limited number of This paper describes the application of MALDI-FTMS to identifiable matrix peaks and these matrix signals should not the structural characterization of the PAH-DE nucleoside be unduly affected by the presence of salts, buffers, and other and nucleotide adducts depicted in Figure 1. Represented in common sample components. The matrix should also remain this group are one deoxyadenosine (l),two deoxyguanosine chemically inert with respect to the analyte in the solution (2, 3), and one deoxyguanosyl 3'-phosphate (4) PAH-DE phase prior to crystallization and during the laser desorption adducts. In each case, the trihydroxy-tetrahydroPAH portion process. Undesirable analyte "photoadducts" (analytes altered of the adduct is covalently attached at the exocyclic amino by additions .of the matrix or decarboxylated or dehydrated group of the adenine or guanine base. Following a general matrix fragment^^^) are often observed in the analysis of high discussionof the experimental conditions that have been found molecular weight proteins, and the formation of such phouseful for MALDI-FTMS, features of the MALDI and CID toadducts would complicate the identification of unknown mass spectra of PAH-DE adducts will be reported. DNA adducts. A final consideration relates to analyte MALDI-FTMS Conditions. The extension of MALDIfragmentation. MALDI-FTMS studies have shown that the matrix affects the extent of desorption- or ionization-induced TOF to FT mass analyzers has required the use of an ion fragmentation of oligonucleotide^.^^ Ideally, the matrix should deceleration technique for the efficient trapping of MALDIprovide a predictable degree of analyte fragmentation, produced ions.36.38 The spectra reported in this paper were permitting additional control over the FTMS experiment. measured using deceleration times (tD) of 50 f 5 ps, although spectra were monitored using a range of deceleration times In thedevelopment of MALDI conditions for the structural to ensure detection of all low-mass fragments and any highcharacterization of nucleoside and nucleotide PAH-DE mass impurities or analyte dimers. For the PAH-DE adducts, adducts, a variety of matrix materials were investigated. The optimal deceleration times provided some discrimination materials chosen for study include three matrices that have worked well for the analysis of proteins, FA, SA,53 and against low-mass matrix ions; however, FTMS ion ejection DHBA.45 We have also evaluated two matrices that have techniques were also employed to eliminate matrix ions from been used successfully for the analysis of oligonucleotides, the cell and improve the dynamic range for detection. HPA4' and a 4:l mixture of AA and NA.43 These matrices For the analysis of biomolecules, the MALDI matrix plays exhibit different characteristics with respect to vacuum an essential role in the desorptionand ionization p r o c e s ~ e s . ~ ~ - ~ ~ sublimation, signal reproducibility, appearance of the crysOf the hundreds of compounds evaluated by researchers, only talline sample, the production of matrix ions, and the degree a few have been identified as efficient matrices, and of these of analyte fragmentation. In the following discussion, an few, certain matrices facilitate the detection of particular attempt will be made to summarize some of the matrix classes of biological materials. For example, DHBA is an characteristics as they pertain to the FTMS analysis of PAHexcellent protein matrix;45 however, this material does not DE adducts. General mass spectral features of the more work as well for the desorption and ionization of oligonuclecommonly used matrices have been described else~here.~5951 o t i d e ~ . For ~ ~the * ~analysis ~ ~ ~ of ~ oligonucleotides, matrices Additional matrix ions were observed in this study, which such as HPA47 and AA-NA43 produce superior signals. resulted from reactions of the matrix with sodium ions present Studies have also shown that the matrix plays an important in the adduct samples. A detailed discussion of matrix ions role in the efficiency of positive vs negative48and singly vs is beyond the scope of this paper; however, as a general recommendation, the identification of interfering matrix peaks (43) Nordhoff, E.; Ingendoh, A.; Cramer, R.; Overberg, A.; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun. Mass Specrrom. 1992.6.771776. (44) Li, Q.M.; Dillen, L.; Claeys, M. Biol. Mass Specrrom. 1992, 21, 408-410. (45) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Specrrom. Ion Processes 1991, I l l , 89-102. (46) Parr, G. R.; Fitzgerald, M. C.; Smith, L. M. Rapid Commun. MassSpectrom. 1992, 6. 369-312. (47) Wu, K. J.; Stding, A.; Becker, C. H. Rapid Commun. MassSpecrrom. 1993, 7, 142-146. (48) Juhasz, P.; Costello, C. E. J. Am. Soc. Mass Specrrom. 1992, 3, 185-796.
(49) Beavis, R. C.; Chaudhary, T.; Chait, B. T. Org. Mass Spectrom. 1992, 27. 156-158. (SO) Beavis, R. C. Org. Mass Specrrom. 1992, 27, 653-659. (51) Ehring, H.; Karas, M.; HiBenkamp, F. Org. Mass Specrrom. 1992,27,472480. (52) Juhasz, P.; Costello, C. E.; Biemann, K. J. Am. SOC.Mass Spectrom. 1993, 4, 399-409. (53) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Specrrom. 1989, 3, 432435.
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is best accomplished by the analysis of sample blanks that contain any buffers or salts found in the actual sample. Matrix Selection: Experimental Considerations. Of the matrices evaluated in this study, DHBA was found to be the most useful. This matrix produces consistent signals for hundreds of laser shots at one location, which facilitates the acquisition of CID spectra. The matrix sublimesslowly under vacuum, and signalsare produced even after leaving the sample under vacuum overnight. In addition, the solid matrix encapsulation of the analyte appears to stabilize the samples toward storage in the laboratory at atmospheric pressure, and PAH-DE adduct samples have provided comparable signals after storage for 2 weeks. DHBA matrix solutions can be prepared at high concentrations (1.5 Min 1:l CH&N-H20), which has theadvantage of producing crystalline needles uniformly covering the probe tip where the matrix-analyte solution was deposited. This feature makes it easy to find the crystalline sample once the probe is inserted into our FTMS instrument, which is not equipped with viewing optics. The high matrix concentration has two drawbacks. First, a larger amount of sample is required to achieve an appropriate matrix to analyte mole ratio. For this study 1 pL of the 20-70 pM adduct solution was generally mixed with 0.5 pL of the concentrated matrix solution, which resulted in the best compromise between dilution of the matrix solution and maintenance of the matrix to analyte ratios below 105. When lower matrix concentrations (0.06 M) were used, samples consisting of a rim of needlelike crystals surrounding a region of granular crystals were produced, a result that has been commonly observed in previous studies.45Crystallizationunderthese conditions has the benefit of requiring less sample and is also thought to provide a degree of sample purification, with salts collecting in the inner region of granular ~ r y s t a l showever, ; ~ ~ ~ ~problems ~ with locating the outer crystalline sample rim on our instrument led us to use larger sample quantities combined with techniques such as the application of ion-exchange beads43or sample desalting procedures to reduce the salt concentration in the analytematrix hixture. Both SA and FA sample preparations resulted in the formation of smaller crystals that were evenly distributed over the sample spot, which made the sample easy to locate on the probe tip. Compared with DHBA, relative abundances of adduct and matrix ions measured using SA and FA were more dependent upon careful adjustment of the laser irradiance, although the degree of adduct fragmentation was unaffected. For CID experiments, the most persistent signals were obtained when the probe was rotated back and forth during data acquisition, although the same sample area was repeatedly illuminated. While all the matrices produced cationized matrix peaks, cationized matrix ions from SA and FA were more problematic because of the number of peaks observed and their appearance at m / z values in the molecular ion and adducted base fragment region of the MALDI mass spectra. These cationized matrix peaks included sodiated molecular ion peaks at [M + nNa - ( n - l)H]+, with n = 0-3, and sodiated dimers at [2M nNa - (n - l)H]+, with n =
0-4. Because of these matrix ions, lower matrix to analyte mole ratios (103-102:1) gave the best results. While the two oligonucleotide matrices (AA-NA and HPA) provided useful adduct mass spectral information, they were difficult to work with experimentally. The mixture of AA and N A rapidly sublimed under vacuum and signals were produced only for 10-15 min. This short time frame limits the number of experiments that could be performed. In some instances, signals could be regenerated by the application of additional matrix material. Matrix ions also proved to be problematic for the AA-NA mixture, especiallyin the negative ion mode, because of the production of complex clusters of ions. HPA sample preparations gave an outer rim of broad crystals with needlelike crystals that extended across the probe tip in some preparations. Because of the sparse coverage of the probe tip and the limitation of working without viewing optics, finding a good spot with this matrix was tedious and it was difficult to generate persistent signals that could be used for CID experiments. Matrix Selection: Mass Spectral Considerations. The ability to ionize biomolecules without fragmentation is one frequently cited advantage of MALDI-TOFMS for the analysis of proteins. For other compound classes, such as oligonucleotide^^^^^^ and gangliosides,48 analyte fragmentation has been observed in TOF studies. In these studies, the MALDI matrix selection influenced the degree of analyte fragmentation. On the FTMS, analyte fragmentation is generally more extensive than that observed on TOF systems.36 This effect may be a consequence of the longer time frame for detection on FTMS vs TOFMS, where FTMS detection requires tens to hundreds of milliseconds, as opposed to the microsecond time frame associated with the TOFexperiments. In addition, the metastable fragmentation of a precursor ion following acceleration in a linear TOFMS would not be as apparent in the mass spectrum because both fragments and the undissociated precursor arrive at the detector at the same nominal time, although peak broadening may occur. In contrast, fragment ions appear at their true m / z values in the FTMS experiment. Because the FTMS experiment is more affected by analyte fragmentation, matrix effects on analyte fragmentation become more apparent, and matrix selection can be used to control the degree of structural information obtained from the direct MALDI mass spectrum. For the analysis of the adducted nucleosides depicted in Figure 1, matrix selection determines the degree of analyte fragmentation. To illustrate this effect, MALDI-FTMS spectra of the dimethylchrysene-(DMC-) DE adducts of deoxyadenosine (1) and deoxyguanosine (2), measured in the five MALDI matrices, are shown in Figures 2 and 3. Major fragment ions and fragmentation pathways (discussed in more detail below) are depicted in Schemes 1 and 2. In this paper, the protonated adducted base (aglycon) fragment is represented by BH2+ and the protonated deoxynucleosidefragments are represented by dAH2+ (deoxyadenosine) and dGH2+ (deoxyguanosine), while AH2+ and GH2+ serve to represent the protonated adenine and guanine base fragments. R+, [R - HzO]+, and [R - H20 - CO]+ denote the positively charged trihydroxy tetrahydro PAH fragment and ions
(54) Wang, B. H.; Dreisewerd, K.; Bahr,U.; Karas, M.; Hillenkamp, F. J . Am. SOC. Mass Spectrom. 1993, 4 , 393-398.
( 5 5 ) Tang,K.;Allman,S. L.; Jones,R. B.;Chen,C.H.;Araghi,S.RapidCommun. Mass Spectrom. 1993, 7, 435-439.
+
1278
Analytical Chemistry, Vol. 86, No. 8, April 15, 1994
A
[R-H20-C0It261
\
80 0
B
152 GHz+ / [R-H20-CO]+
800
,;:*:..;.,,I,, ,
B
,
,
,[
,
,,
,
261 /307
,
,A
MH+ 574
200 00
1 :
BH*+ 200
x 00
1000
1
800 400 20 0
x 2000
80 0
E
3000
1000
5 w O
BWO
mi*
00
1000
2000
lWO
3000
6000
WOO
7000
mi2
Flguro 2. Positive ion MALDI-FT mass spectra of e(deoxyadenosinIIP-yl~B,~methyCl,Z,3.trihydoxy-l,2,3,4-tetrahydrochrne (1) using (A) sinaplnlc acM, (B) ferulic acM, (C) 2,5dihydroxybenzoic acM, (D) anthranllic acid-nicotinic acM, and (E) 3-hydroxyplcollnic acM as the MALDI matrix. A total of 20 ng of adduct 1 applied: matrix to analyte mole ratio varied from lo3:1 to lo4:1. Ion-exchange beads applled for (B-E). Matrix ions indicated by X.
Flguro 3. Positlve ion MALDI-FT mass spectra of qdeoxyguanosinI\eyl)s,~imethyC1,2,3.trihyctoxy-l,2,3,4-tetrahydrochrysene (2) using (A) slnapinlc acid, (B) ferullc acid, (C)2,5dihydroxybenzoic acld, (D) anthranilic acid-nicotinlc acid, and (E) 3-hydroxypicollnic acid as the MALDI matrix. A total of 40 ng of adduct 2 applied; matrix to analyte mole ratio varied from 104:1to 10s:l. Ion-exchange beads applied for (B-E). Matrix Ions Indicated by X.
produced by subsequent neutral losses of H20 and CO (see Schemes 1 and 2). In general, SA, FA, and DHBA (the three protein matrices) all promote fragmentation of the ionized DMC-DE adducts, while fragmentation is reduced for spectra measured using AA-NA, and the least fragmentation is induced by HPA. Within the group of protein matrices (SA, FA, DHBA), SA shows the highest [BH2 - H~O]+/[BHZ]+and [R - H2O CO]/[R - HzO]+ ion ratios, indicating that more extensive sequential decompositions have occurred. These ratios are lower in the spectra measured with AA-NA and HPA, where primary fragmentations from MH+ are also reduced. Under MALDI conditions, factors that may influence the degree of analyte fragmentation include the energy imparted during the desorption or ionization process and analyte cooling in the expanding plume. The variations in the degree of PAHDE adduct fragmentation, as determined by the matrix selection, call to mind fragmentation variations induced under CI conditions by controlling the exothermicity of protonation reaction^.^^,^^ While the mechanisms of ionization under
MALDI conditions are still under investigation, evidence exists supporting the role of gas-phase protonation and cationization reactions.54 If some broad assumptions are made regarding proton-transfer reactions under MALDI conditions, and we consider ionization to result by proton transfer from the protonated form of the matrix, a rough correlation is found between the degree of fragmentation and the proton affinity (PA) of the most basic site on the matrix molecule. For example, PA values (in kcal mol-') increase from phenol (196) < anisole (200) < aniline (21 1) < pyridine (220),56 and this increase in the PA of the most basic siteon the matrix parallels the reduction in analyte fragmentation observed as one changes from the methoxy/hydroxy-substitutedaromatic acid matrices (SA, FA, DHBA), to 2-aminobenzoic acid (the dominant ionized matrix component in the AA-NA spectrum), to the substituted pyridine, HPA. Alternatively, the analyte fragmentation variations may reflect desorption-related phenomena, with the matrix selection influencing intermolecular matrix-analyte interactions and the mechanisms of energy
(56) Harrison, A. G . Chemical Ionization Mass Spectrometry; CRC Press: Boca Raton, FL, 1983; p 33. (57) Wilson, M. S.; McCloskey, J. A. J . Am. Chem. SOC.1975, 97, 34363444.
(58) Mowry, C. D.; Johnston, M. V. Proceedings of the 40th ASMS Conference on MassSpectrometry and Allied Topics, Washington, DC, May 3 I-June 5, 1992; p 364. (59) Claereboudt, J.; Clacys, M.; Gcisc, H.; Gijbcls, R.; Vertes, A. J . Am. Soc. Mass Specrrom. 1993, 4, 798-812.
Analyrical Chemistry, Vol. 66, No. 8, April 15, 1994
1270
Scheme 1
.
J
dZ558
I
mh136
I
Scheme 2
H+ or Na*
H+ or Na'
mh 290 (dGHNa+)
ii (+H)
mh152 (GH:) mh 174 ( G H N ~ )
*
-H20
HO
OH
/
HO
m/z 574 (MH')
mh 440
[BHz-2H20It mh 422
c @ OH
mh 596 (MNa*)
ii/
-H20
__t [BHZ-H20]'
mh458 (BH:) mh480 (BHNa*)
-co [R-H20-COIt + -H,O [R-H,O]' +
HO
HO
mh 289
/ OH
mh 261
mh307 (R')
transfer. Studies of neutrals have shown that internal energies are influenced by the matrix and i r r a d i a n ~ e . ~ * , ~ ~ On a practical note, the reduction of fragmentation when HPA and AA-NA are used provides useful information about sample purity, while the protein matrices (SA, FA, DHBA) 1200
Analytical Chemlstty, Vol. 66, No. 8, April 15, 1994
provide more structural information than is typically available under FAB-MS conditions. Accurate Mass Measurements. Using DHBA as the matrix to promote analyte fragmentation, accurate mass measurements were made for the DMC-DE adducts 1 and 2. For the
Table 1. Exact Mare Measurements for 4-(Deoxyadenodn~-yl)5,6dlmathyl-1,2,3-trlhydroxy-l,2,3,4-tetrahydroch~~ne(1) Udng DHBA as the MALDI Matrix'
ion
mass (Da) calcd measd
formula
CmH320& CzsHuOsNs C&zOzN6 CzoHleOa R+ C20H1702 [R - HzOl+ [R - HzO - CO]+ C1SH1,O Cl&l&Ns dAH2+ MH+ BH2+ [BHz - HzOl+
558.234 71 442.187 37 424.176 80 307.132 87 289.122 31 261.127 39 252.109 12
558.230 45 442.187 11 424.178 09 307.133 49 289.123 38 261.127 24 252.109 39
difference (ppm) -7.6 -0.6 3.0 2.0 3.7 -0.6 1.1
a S ectra were calibrated using ions from the internal calibrant, d(3’-8T-5’), and the AH2+ ion from adduct 1. Resolution was 6K for MH+ and 14K for dAHZ+.
Table 2. Exact Mass Measurements for 4-(Deoxyguanosln~-yl)b,6dlmethyCl,2,3-trlhydroxy-l,2,3,4-tetrahydrochrysene (2) Udng DHBA as the MALDI Matrix'
ion MNa+ BHNa+ BH2+ [BHz-HzO]+
formula
calcd
CmH3101NsNa CdmOdNsNa Cdu04N5 CdzaOsNs CZOH1903
596.211 57 480.164 22 458.182 28 440.171 72 307.132 87 289.122 31 261.127 39 290.085 97 174.038 63
R+ [R- HzOl+ Cd1102 [R- HpO - COl+ ClgH170 dGHNa+ GHNa+
Cl,&O4NdVa C6HSON6Na
(Da) measd 596.202 43 480.161 44 458.178 75 440.171 04 307.129 78 289.120 55 261.126 06 290.086 01 174.039 54
AI
difference (ppm) -15.3 -5.8 -7.7 -1.5 -10.1 -6.1 -5.1 0.1
5.2
a S ectra were calibrated using ions from the internal calibrant, d(3’-8TT-5’),and the GH2+ ion from adduct 2. Resolution was 4K for [M + Nal+ and 13K for dGHNa+.
mass assignments reported here, d(3’-CT-5’) was used as the calibrant along with the protonated base fragment (either GH2+ or AH29 from the ionized adduct. The calculated and measured masses for adducts 1 and 2 are listed in Tables 1 and 2, respectively, and these assignments are consistent with the elemental compositions of the fragment ions depicted in Schemes 1 and 2. Resolutions were in the range of 4-18K fwhm. Initial attempts to use rhodamine 6G or rubrene as internal calibrants were unsuccessful. While signals for these calibrants were detected, they were produced at locations on the crystalline sample where no adduct signals were observed, suggesting that the calibrants were not uniformly incorporated in the adduct-matrix mixture. In contrast, after adding either 2’-deoxyadenosine, 2’-deoxyguanosine, or d(3’-CT-5’) as internal calibrants, a single laser shot gave signals for both the adduct and internal calibrant. Positive Ion MALDI and CID Mass Spectra of PAH Adducts. As briefly described above, the direct MALDI-FT mass spectra of PAH-DE adducts can provide both molecular weight information and structurally relevant fragment ions, with the degree of fragmentation controlled by selection of an appropriate matrix. In this section, we will describe these fragmentation pathways in more detail and show how structural information obtained directly from a MALDI mass spectrum may be augmented through the use of CID experiments. Spectra of the methylchrysene- (MC-) DE deoxyguanosine adduct 3 and the BaP-DE guanosyl 3‘phosphate adduct 4, both measured using DHBA as the matrix, are shown in Figure 4A,B. MALDI mass spectra of adduct 3 were also measured using all five MALDI matrices. The
200
I
I l l 1000
2000
,
3000
I 4000
I 5000
6000
7000
mir
Flgure 4. Positive Ion MALDI-FT mass spectra: (A) e(deoxyguanosinhP-yl)5-methyC1,2,3tihydroxy-1,2,3,4-tetrahydrochrysene (3),with 30 ngof adduct applied following sample desaMng; (E) 7R,lS$Strihydroxy1OR-(hPdeoxyguanosyl 3‘-phosphate)7,8,9,lO-tetrahydrobenzo [a] pyrene (4), with less than 25 ng applied with ion-exchange beads. DHBA was the MALDI matrix.
-
variations in fragmentation of 3 paralleled those observed for 2, with the protein matrices, SA, FA, and DHBA, inducing the most extensive fragmentation. The nucleotide adduct 4 was also desorbed from the AA-NA matrix. In this study the potential of MALDI-FTMS for the structural characterization of PAH-DE adducts was evaluated, and no attempt was made to determine the ultimate sensitivity of this technique; however, the sample quantities used in this study, 1 0 4 0 ng (20-70 pmol) applied, are much lower than those required for the direct FAB analysis of underivatized adducts.20*21In addition, the MALDI-produced signals are long-lived compared with signal generated by FAB. When DHBA was used as the matrix, one sample application could easily be used for mass spectral measurements and extensive CID experiments requiring hours to complete. In general, the experiments were completed long before the sample was consumed. Future strategies for lowering detection limits will involve the application of smaller volumes of the adductmatrix mixture to the probe and optimization of the adduct to matrix ratio. Protonated and Cationized Molecular Ions. The DMCDE deoxyadenosine adduct 1 gave abundant protonated molecular ions at m / z 558 in all the matrices examined in this study (see Figure 2). Although sodium was present in these samples, cationized molecular ions at [M + Na]+ were weak or absent. CID of the MH+ ion from 1 gave fragment ions (BH2+, [BH2 - H20]+, [R - H20 - CO]+, dAH2+, AH2+) that duplicated information provided by the direct MALDI spectra measured using SA, FA, and DHBA. In the case of the DMC and MC-DE adducts of deoxyguanosine (2, 3), much weaker protonated molecular ion signals were observed (see Figures 3 and 4A). The weaker MH+ signals may result from more extensive fragmentation of the protonated molecular ion, as evidenced by the abundance of the adducted base, BH2+, and other fragments (GH2+ and [R - H2O CO]+) in the spectra. Even when HPA is used as the matrix, the BH2+ fragment appears as the base peak in the spectrum. Because the deoxyguanosyl MH+ ion signals in the protein Analytical Chemistty, Vol. 66,No. 8, April 15, 1994
1201
1000
[MtNa]'
~A
80 0 -
BHNa+
596
-
6951
[MtNa]+ 596
480
1
I
I
A
\ BHNa*
5
5 347i
GHNa+
174
00
dGHNa'
I
2000
1000
I
29"/
4000
3000
5WO
i I
6000
mlz
16 9
00
Figure 5. Positive ion MALDI-FT mass spectra of e(deoxyguanosinff-yl)-5,6dimethyC1,2,3-trihydroxy-l,2,3,4-tetrahydrochrysene (2) in (A) DHBA, using 40 ng of adduct applied and no sample desatting or ion-exchange beads applied. (B) MALDI-FT mass spectrum of 2 with 40 ng of adduct applied and LiCl added to the DHBA matrix at a 2: 1 mole ratio.
matrices were weak, and because of difficulties in working with HPA, no CID spectra of the MH+ ions from 2 or 3 were measured. The BaP-DE guanosyl 3'-phosphate adduct 4 produced the weakest molecular ion signals in the positive ion mode (see Figure 4B). These results are consistent with previous studies of the laser microprobe desorption of nucleosides and nucleotides,m where protonated molecular ions were only observed for adenosine and deoxyadenosine. While the deoxyguanosyl adducts 2 and 3 gave weak protonated molecular ion signals, these compounds easily formed [M Met]+ ions, where Met represents a metal ion such as Na+, Li+, or Cu+. Cationized molecular ions have been observed in other laser desorption studies of nucleoDue to the presence of sodium ions in the samples examined in this study, sodiated forms of both the molecular ion and base-containing fragments were commonly observed unless steps were taken to removesodium. The deoxyguanosyl adducts also showed a propensity toward cationization with copper, when this ion was present as an impurity in the sample or matrix solution. The spectra shown in Figure 3B-E were measured after ion-exchange bead or desalting techniques were used to reduce the sodium ion concentration. Without these salt removal techniques, the spectrum measured using DHBA was dominated by [M Na]+ ions (see Figure 5A), and [M + 2Na - H]+ ions were also observed. Analysis of the deoxyguanosyl adducts presents a dilemma regarding the best conditions to provide both molecular weight and structural information. When the protein matrices (SA, FA, DHBA), which provide sufficient fragment ion signals, were used, MH+ signals were weak, and the abundance of [ M + Na]+ ions and associated sodiated fragments depended upon the amount of sodium present in the sample. While MH+ ions were produced when HPA was used as the matrix, the spectra provide insufficientinformation regarding the PAH triol portion of the molecule, and the use of CID to enhance
+
+
(60) Van Vaeck. L.; Van Espcn, P.; Adam, F.;Gijbels, R.; Lauwers, W.; Esmans, E. Biomed. Enuiron. Mass Specrrom. 1989, 18, 581-591. (61) Chiarelli, M. P.; Gross,M. L.J. fhys. Chem. 1989, 93, 3595-3599. (62) McCrery, D. A.; Gross, M. L. Anal. Chim. Acra 1985, 178, 91-103.
1282
Analytical Chemistry, Vol. 66,No. 8, April 15, 1994
1000
2000
3000
4000
5000
6000
7000
mir
Figure 8. Positive ion CID mass spectra: (A) [M 4- Na]+ ion of adduct 2, mlz 596; spectrum prior to ion Isolation shown in Figure 5A. (B) [M Li]+ ion of adduct 2, mlz 580; spectrum prior to ion isolation
+
shown in Figure 5B.
structural information was difficult to implement on a practical basis using either HPA or AA-NA. In an attempt togenerate consistent [M Met]+ ion signals while retaining structurally informative fragments, NaCl or LiCl was added to the DHBA matrix-analyte mixture at a 1:2 mole ratio of matrix to metal salt. A typical spectrum produced upon addition of the lithium salt is shown in Figure 5B. The addition of NaCl at a 1:2 mole ratio gave a spectrum similar to that shown in Figure 5A, although sodiated peaks appeared at higher abundances relative to the protonated ions and PAH triol fragments. The addition of either sodium or lithium chloride resulted in the production of abundant cationized molecular ions, along with cationized forms of the base (BHMet+), deoxyguanosine (dGHMet+), and guanine (GHMet+). While cationization facilitated identification of the molecular ion, fragment ions associated with the PAH triol portion of the molecule (R+, [R - H20]+, [R - H20 CO]+) were attenuated. Isolation and CID of the sodiated or lithiated molecular ions resulted in production of thesodiated adducted base, BHNa+, and the lithiated or sodiated deoxyguanosine nucleoside and guanine base (see Figure 6A,B). Cationization is often used to alter fragmentation pathways because of differences in the site of H+ vs Met+ interaction.63 For the PAH-DE adducts studied here the metal ion appears to be more strongly associated with the guanine base, possibly through coordination at the carbonyl group, and fragmentation is redirected with charge being retained on thedeoxyguanosine and guanine fragments. Redirecting fragmentation in this manner reduces information related to the PAH triol. These results are similar to those observed in FAB MS/MS studies of MH+ and [M + K]+ ions from adducts of pyrrolizidine alkaloid metabolites, where [M + K]+ gave only products with K+ associated with the base or nu~leoside.~4 Protonated and Cationized Adducted Base Ions. The DMC-DEdeoxyadenosine adduct 1shows an abundant BH2+ fragment ion at m/z 442 in the MALDI-FT mass spectrum (see Figure 2), which results from cleavage of the glycosidic
+
(63) Gas-Phase Inorgonic Chemistry; Russell, D.H., Ed.; Plenum: New York, 1989. (64) Tomer, K. B.;Gross, M. L.; Deinzer, M. L. Anal. Chem. 1986,58,2527-2534.
OH
-AH
CH3
-co 0 CH3
CH3 [R-H20-CO]+
[R-H20]*
mh 261
mh 289
bond with transfer of one sugar hydrogen back to the base. This loss of the neutral deoxyribose sugar is a characteristic fragment appearing in the EI, CI, and FAB mass spectra of nucleosides.' Associated with the BH2+ fragment are losses of one and two molecules of water, dehydrations that may serve to aromatize the trihydroxy-substituted ring. The BH2+ fragment and associated water losses are also observed in spectra of the deoxyguanosyl adducts 2 and 3 and, to a lesser extent, in the spectrum of the BaP guanosyl 3'-phosphate adduct 4 (see Figures 2-4). When FAB-MS/MS is used for the characterization of DNA adducts, CID of the BH2+ion is often used for structural characterization.20p21Isolation and CID of the BH2+ fragment from the deoxyadenosyl adduct 1 results in production of the protonated adenine base, AH2+, the PAH triol fragments, [R - H2O - CO]+ and [R - HzO]+, and the [BH2 - H20]+ ion (see Figure7A). Fragmentationtogive the [R-HzO-CO]+ product may occur via the route shown in Scheme 3. When the BH2+ ion from 2 is subjected to CID, similar products are produced; however, the protonated guanine base, GH2+, is - 4 times more abundant than the [R - H2O - CO]+ PAH triol fragment. This behavior parallels that observed in the direct MALDI mass spectra, where the protonated base fragment dominates the mass spectrum. Fragmentation of the protonated adducted base to give the GH2+ ion involves hydrogen transfer from the PAH triol. Isolation and CID of the dehydrated adducted base ions [BH2 - HzO]+ from compounds 1 and 2 show only an additional loss of H2O and fragmentation to give the protonated base ions AH2+ and GH2+, respectively. The absence of PAH triol fragments suggests that dehydration reduces the proton affinity of the [R - HzO] neutral relative to R. Cationized adducted base fragments were observed in the spectra of guanosyl adducts 2 and 3 when sodium or lithium were present in the matrix. For adduct 2 these ions appear at m / z 480 and 464, respectively (see Figure 5A,B). Isolation and CID of these cationized adducted base fragment ions gave the cationized base ions GHNa+ and GHLi+,respectively, as the sole fragments (see Figure 7B,C). As was discussed above, cationization alters the fragmentation patterns by
__
: A
D
'
BH2+ I' i4 2
~
54 73
\
4101
13 7
00
500 i
1000
1500
2000
2500
3000
4000
3500
\I
BHNa'
GHNa' 174
\
5561
4500
5000
1
480
I"
1wo
200 0
300 0
22371
5
1
\I '
BHLi' 464
GHLi+
1678
5
e
500 0
400 0
. ..
11 19-
1000
200 0
300 0
mil
400 0
500 0
Figure 7. Positive ion CID mass spectra: (A) BH2+ ion of adduct 1, m/z442; spectrum prior to ion isolation shown in Figure 2C. (B) BHNa+ ion from adduct 2, mlz 480; spectrum prior to ion isolation shown in Figure 5A. (C) BHLi+ ion of adduct 3, m/z 464; spectrum prior to isolation shown in Figure 58.
charge retention on the base. Thus, cationization is undesirable from the standpoint of providing more structural information about the PAH triol adduct. P A H Triol Fragments R+,[R- H20]+, and [R- H20 CO]+. Ions providing structural information regarding the adducted PAH-DE appear at R+, [R - HzO]+, and [R - HzO - CO]+,as shown in Schemes 1-3. Analogous fragmentations have been observed in the CID mass spectra of FAB-generated protonated BaP-DE adducts of adenine, guanine, and 2'deoxyadenosine.22 The dehydration and loss of CO appear to be particularly facile processes once R+ has been liberated Analytlcal Chemistty, Vol. 66, No. 8, April 15, 1994
i283
56 7
-53 2461
IA
Scheme 4
m
231
14 2
/
mh 239
OH [R-H20-CO]* mh 257
I 200
I
35012 0
I
in
: e
21 07
1405
2
n-I
IYII I
1
2391 257 21 5 l e i - H "
1
[R-H20-CO-C2H20It
mh 215
I IPI
1
l ~
, 7 02
0 00 0
-
-
Figure 8. Positive ion CID mass spectra: (A) [R H20 CO]+ ion of adduct 1, mlr 261; spectrum prior to ion isolation shown In Figure 2C. (8)[R H20- CO]+ ion of adduct 3, mlz 247;measured using FA as the matrix. (C)[R H20 - CO]+ ion of adduct 4; m/z 257; spectrum prior to ion isolation shown in Figure 48. (D) [R - H20]+ ion of adduct 4; mlz 285;spectrum prior to ion isolation shown in Figure 48.
-
-
from the nucleoside, as evidenced by the low abundance of the R+ peak. While the R+, [R- H2O]+,and [R-H20- CO]+fragments are characteristic of the PAH triol moiety, they provide little information regarding the identity of the PAH and ring substituents. Additional information is provided by isolation and CID of the [R - H2O - CO]+ ion. CID mass spectra of the [R - H2O - CO]+ ion (mlz 261) from the DMC-DE adducts 1 and 2 are essentially identical, showing products resulting from the loss of one and two methyl radicals. The CID mass spectrum of m/z 261 from 1is shown in Figure 8A. The CID mass spectrum of the m/z 247 ion from the DM-DE adduct 3 is shown in Figure 8B. This spectrum shows a single product resulting from loss of one methyl radical. Thus the CID spectrum of the PAH triol fragment provides additional information regarding the number and type of ring substituents. In the case of the BaP-DE adduct 4, isolation and CID of the m / z 257 ion results in formation of two dominant fragments at m/z 239 and 215. The m/z 239 ion is produced by loss of H2O (see Scheme 4) while the m/z 215 ion may be produced by net loss of C2H20 from [R - H2O - CO]+ to give the ring-expanded tropylium-type structure shown in Scheme 4. CID of the [R - HzO]+ ion from the BaP adduct 4 gives the m/z 257 fragment by loss of CO and shows fragments similar to those observed in the CID mass spectrum of m/z 257 (see Figure 8D). These spectra show that CID 1284
Analytical Chemistty, Vol. 66, No. 8, April 15, 1994
of MALDI-produced fragments provides additional information that may help characterize the PAH triol moeity. Protonated and Cationized Nucleoside and Base Fragments. The ions appearing at m/z 252 and 136 in the spectrum of 1 correspond to protonated adenine, AH2+, and deoxyadenosine, dAH2+, respectively (see Scheme l), and serve to identify the nucleoside portion of the adduct. Detection of the protonated nucleoside has been associated with covalent attachment of the diol epoxide adduct at the exocyclic amino group from FAB-MS/MS studies.22 The guanosyl adducts showed abundant protonated base fragments, GH2+, at m/z 152; however, the protonated nucleoside was often weak or not observed. In contrast, cationization of the adduct resulted in production of both the cationized base and nucleoside (see Figures 3 and 5), suggesting that cationization stabilizes the nucleoside with respect to fragmentation to the cationized base. [ M +3H]+ Zons. While the MALDI mass spectra showed no evidence for additions of intact, decarboxylated, or dehydroxylated forms of the matrix to the PAH-DE adducts, the DHBA spectrum of the deoxyadenosine adduct 1did show ions resulting from hydrogen additions. In the molecular ion region an [M + 3H]+ ion is observed, which fragments to give an m/z 254, a reduced form of 2'-deoxyadenosine ([dA 4H] +). The m/z 254 ion is also observed in the MALDI mass spectrum of 2'-deoxyadenosine, measured using the DHBA matrix. Ions 2 Da above the MH+ ion have been observed in the CI mass spectra of some unsaturated pyrimidine nucleosides and in the spectrum of N6-(3-methy1-2-butenyl)adeno~ine.5~ Hydrogen additions have also been observed in the positive ion FAB mass spectra of nucle0sides6~and nucleotides66and in the negative ion FAB mass spectra of oligonucleotides.67 Under MALDI conditions, hydrogen additions have been observed in the mass spectra of matrix compounds, and these additions have been attributed to reactions of hydrogen radicals in the MALDI plume,51and [M 3H]+ and [M + HI- ions have been detected in the MALDI-FTMS mass spectra of
+
+
(65) Cerny, R. L.;Gross, M.L. AMI. Chcm. 1985, 57, 1160-1163. (66) Fenselau, C. J. Not. Prod. 1984, 47, 215-225. (67) Laramb, J. A.; Arbogast, B.;Deinzer, M.L.AMI. Chem. 1989, 61, 21542160.
1000
E
[ M-H-PHpOI-
A
80 0
[M-HI'
20 0
i -
1
losses of H2O were observed in the spectrum, possibly due to deprotonationof the 3'-phosphateand a change in ion internal energy or charge localization. For the guanosyl adducts, the negative ion mass spectra provide information that complements the positive ion spectra. For example, when AA-NA is used as the matrix, the positive ion spectra provide information on the PAH triol, the adducted base, and the identity of the nucleic base, while the negative ion spectra provide molecular weight information.
moj
[M-H-SHpO]'
ii [M-HI'
IC
80 04
6491
346
I /
Ftgurr 9. Negative ion MALDI-FT mass spectra of (A) qdeoxyguanosln-hP-yl)5,&dlmethyC1,2,3-trihydroxy-l,2,3,4-tetrahydrochrysene (2) with 40 ng of adduct applied, (B) 4-(deoxyguanosln-Wyi) 5-methyC1,2,3-trlhydroxy-l,2,3,4-tetrahydrochrysene (5) with 10 ng of adduct applied, and (C) 7R,8S,9Strlhydroxy-lOR(Mdeoxyguanosyl 3'-phosphete)-7,8,9,1O-tetrahydrobenzo[a]pyrene (4) with less than 25 ng of adduct applied, using AA-NA as the MALDI matrix.
small oligonucleotide^.^^ In FAB and secondary ion mass spectrometric studies, the detection of hydrogen additions has been explained by reduction reactions in the solid or liquid matrix, hydrogen atom abstractions from the matrix, or the reduction of multiply protonated ions in s 0 l u t i o n . 6 ~While ~~ our observations lend no new insights into the mechanism by which these ions are formed, the production of ionized nucleic acid components altered by hydrogen additions seems to be common to a number of ionization techniques. Negative Ion MALDI Mass Spectra of PAH Adducts. While the DMC-DE deoxyadenosine adduct 1 gave strong signals in the positive ion mode, no useful signals were detected in the negative ion mode using FA, SA, DHBA, or the AANA mixture. In contrast, negative ion signals were observed for the guanosyl adducts 2 and 3 when the AA-NA matrix mixture was used. The spectra show [M - HI- ions, with fragments resulting from the loss of two molecules of H2O (see Figure 9A,B). The guanosyl3'-phosphate adduct 4 gave a strong [M - HI- ion in the negative ion mode when either DHBA or AA-NA was used as matrix (see Figure 9C). No (68) Detter, L.D.; Hand, 0.W.; Cooks,R. G.; Walton, R. A. MussSpectrom.Rev. 1988, 7,465-502.
CONCLUSIONS The structures of PAH-DE adducts can be characterized using MALDI-FTMS. In the positive ion mode, full MALDI mass spectra, accurate mass determinations, and CID experiments can be implemented using tens of nanograms (20-70 pmol) of sample. MALDI marix selection can be used to control the degree of analyte fragmentation. Two matrices used for oligonucleotideanalysis, AA-NA and HPA, reduced the degree of adduct fragmentation and could be used for molecular mass determination, while three matrices (SA, FA, DHBA) produced protonated (adenosyl adduct) or sodiated (guanosyladducts) molecular ions, accompanied by abundant fragment ions that provide information about the major structural units of the PAH-DE adduct. Accurate mass determinations and CID techniques permitted detailed structural probing of MALDI-produced fragments. Compared with FAB or FAB-MS/MS for the analysis of PAH-DE adducts, MALDI-FTMS permits implementation of accurate mass and CID experiments using lower quantities of sample, and with proper matrix selection, the direct MALDI-FT mass spectra show more structurally informative fragments than are observed under FAB conditions. ACKNOWLEDGMENT The authors thank the American Health Foundation for providing the samples used in this study and J. Caton for performing HPLC analysis of the adduct samples. Research sponsored jointly by the National Cancer Institute under Interagency Agreements DOE0485-FO53-Al and NCIYO1CP-205 12-13, the Office of Health and Environmental Research, US.Department of Energy under Contract DEAC05-840R21400 with Martin Marietta Energy Systems, Inc., and the DOE Faculty Research Participation Program under Contract DE-AC05-760R00033 between the US. Department of Energy and Oak Ridge Institute for Science and Education. Recelved for review October 29, 1993. Accepted January 17,
1994." Abstract published in Advance ACS Absrrucrs, March 1, 1994.
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