Method for the rapid detection and molecular characterization of DNA alkylating agents by MALDI-TOF mass spectrometry

Article abstract of DOI:10.1021/ac101568h

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) may cause DNA adduct formation. While these are commonly detected by the ³²P-postlabeling assay, this method is not informative on the chemical nature of the alkylating agent. Here we report a simple and reliable method that employs MALDI-TOF-MS with 2,5-dihydroxybenzoic acid (DHB) matrix layer (ML) sample preparations for the detection and structural characterization of PAH-DNA adducts. The method involves the enzymatic digestion of DNA to 2′-deoxynucleotides followed by solid phase extraction to remove salt and other contaminants prior to MALDI-MS analysis. By collision induced dissociation (CID) structurally relevant fragments are obtained to permit characterization of the alkylating molecules and the adducted nucleotide. Next to guanosine, adenosine and cytidine adducts formed from reactions with (±)-anti- benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) are identified at a sensitivity of <100 fmol and a mass accuracy of <10 ppm. Studies with (±)-anti-benzo[c]chrysene-9,10-diol-11,12-epoxide (B[c]ChDE) further document the versatility and usefulness of the method. When compared with the ³²P-postlabeling assay MALDI-MS only indentified deoxycytidine as well nucleoside and dinucleotides adducts. Therefore, this sensitive method enables molecular specification and characterization of adducted nucleotides and of the alkylating agent, and thus, provides comprehensive information that is beyond the ³²P-postlabeling assay.

Full text of DOI:10.1021/ac101568h

Anal. Chem. 2010, 82, 8573–8582
Method for the Rapid Detection and Molecular
Characterization of DNA Alkylating Agents by
MALDI-TOF Mass Spectrometry
Ignazio Garaguso,†,‡ Roman Halter,† Jacek Krzeminski,§ Shantu Amin,§ and Ju¨ rgen Borlak*,†,‡
Department of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine,
Hannover, Germany, Center for Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany, and
Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033
Metabolic activation of polycyclic aromatic hydrocarbons
(PAH) may cause DNA adduct formation. While these are
commonly detected by the 32P-postlabeling assay, this
method is not informative on the chemical nature of
the alkylating agent. Here we report a simple and
reliable method that employs MALDI-TOF-MS with
2,5-dihydroxybenzoic acid (DHB) matrix layer (ML)
sample preparations for the detection and structural
characterization of PAH-DNA adducts. The method
involves the enzymatic digestion of DNA to 2′-deoxy-
nucleotides followed by solid phase extraction to
remove salt and other contaminants prior to MALDI-
MS analysis. By collision induced dissociation (CID)
structurally relevant fragments are obtained to permit
characterization of the alkylating molecules and the
adducted nucleotide. Next to guanosine, adenosine
and cytidine adducts formed from reactions with (()-
anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE)
are identified at a sensitivity of <100 fmol and a mass
accuracy of <10 ppm. Studies with (()-anti-benzo[c]-
chrysene-9,10-diol-11,12-epoxide (B[c]ChDE) further
document the versatility and usefulness of the method.
When compared with the 32P-postlabeling assay MALDI-
MS only indentified deoxycytidine as well nucleoside
and dinucleotides adducts. Therefore, this sensitive
method enables molecular specification and charac-
terization of adducted nucleotides and of the alkylating
agent, and thus, provides comprehensive information
that is beyond the 32P-postlabeling assay.
damage events per day. While DNA repair enzymes remove DNA
adducts efficiently, nonrepaired lesion may cause mutations to
initiate malignant transformations.1Thus, exposure to PAH has
been linked to lung, skin, and bladder cancer.2-4 Furthermore,
in molecular epidemiological studies DNA adducts are used to
evidence exposure to hazardous chemicals and to estimate risks
in particular work places or environments.5,6 Notably, PAH are
activated by three major mechanisms involving either one-electron
oxidation to form intermediate radical cations, a reactive o-quinone
and the oxidation to dihydrodiol epoxide (DE) intermediates.7 The
DE formation requires an initial metabolic activation of PAH,
which is efficiently catalyzed by the combined action of cyto-
chrome P450 1A1 and 1B1 resulting in the oxidation of a double
bond to yield an epoxide. The epoxide is then converted to trans
dihydrodiol by microsomal epoxide hydrolase, which undergoes
biotransformation by cytochrome P450 monooxygenase resulting
in an oxidation at the adjacent double bond to generate the
ultimate reactive DE species.8 Such metabolic activation may lead
to sterically hindered bay or fjord PAHDE, which are electrophiles,
that covalently react by cis or trans addition with the exocyclic
amino group of the DNA bases. Studies on PAH metabolism, DNA
binding, mutagenicity and cell transformation assay demonstrated
that PAH such as benzo[a]pyrene (B[a]P), chrysene, benzo[c]-
chrysene (B[c]Ch), benzo[c]phenantrene (B[c]Ph), 5-methyl-
chrysene, dibenzo[a,l]pyrene are activated by this pathway7,9 and
react with amino groups of the purine bases as preferential
alkylating sites.10 Typical targets in DNA are the N2 exocyclic
amino group of guanine, the N6 of adenine and to a lesser
extend the N4 exocyclic amino group of cytosine.11The molec-
ular structure of PAH is an important determinant of their
(1) Dipple, A. Carcinogenesis 1995, 16, 437–41
(2) Schoket, B.; Poirier, M. C.; Mayer, G.; Torok, G.; Kolozsi-Ringelhann, A.;
Bognar, G.; Bigbee, W. L.; Vincze, I. Mutat. Res. 1999, 445, 193–203
.
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous envi-
ronmental pollutants and are primarily formed by incomplete
combustion of organic matter, such as automobile exhaust
emissions, tobacco smoke, coal tar, but are also generated by
cooking of food that has been charred or grilled. While PAH are
not carcinogenic per se, metabolic activation leads to intermediates
that might react with nucleic acid and proteins leading to DNA,
RNA, and protein adducts. Estimates suggest up to 50 000 DNA
.
(3) Bostrom, C. E.; Gerde, P.; Hanberg, A.; Jernstrom, B.; Johansson, C.;
Kyrklund, T.; Rannug, A.; Tornqvist, M.; Victorin, K.; Westerholm, R.
Environ Health Perspect. 2002, 110, 451–88
(4) Phillips, D. H. Mutat. Res. 1999, 443, 139–47
(5) Dipple, A.; Khan, Q. A.; Page, J. E.; Ponten, I.; Szeliga, J. Int. J. Oncol.
1999, 14, 103–11
(6) Peltonen, K.; Dipple, A. J. Occup. Environ. Med. 1995, 37, 52–58
(7) Xue, W.; Warshawsky, D. Toxicol. Appl. Pharmacol. 2005, 206, 73–93
(8) Shimada, T.; Fujii-Kuriyama, Y. Cancer Sci. 2004, 95, 1–6
(9) Amin, S.; Lin, J. M.; Krzeminski, J.; Boyiri, T.; Desai, D.; el Bayoumy, K.
Chem. Res. Toxicol. 2003, 16, 227–31
(10) Dipple, A. IARC Sci. Publ. 1994, 107–29
(11) Cheng, S. C.; Hilton, B. D.; Roman, J. M.; Dipple, A. Chem. Res. Toxicol.
1989, 2, 334–40
.
.
.
.
.
.
* To whom correspondence should be addressed. Phone: +49-511-5350-559.
Fax: +49-511-5350-573. E-mail: borlak@item.fraunhofer.de.
† Fraunhofer Institute of Toxicology and Experimental Medicine.
‡ Hannover Medical School.
.
.
§ Penn State College of Medicine.
.
10.1021/ac101568h  2010 American Chemical Society
Published on Web 09/24/2010
Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 8573

biological activities,12 as the metabolically activated epoxides
located either in a bay or fiord region display different
carcinogenic properties. For instance, the tumorigenic activity
adducts formed upon the reaction of B[a]PDE with naked DNA,
or cell lines and tissue extract of mice that have been exposed to
alkylating agents.27-29 Nonetheless, these methodologies have
some limitations, for example, large amounts of sample require-
ment, extensive liquid-liquid extraction, time-consuming HPLC
or chromatographic purification, as well as expensive instrumenta-
tion. Furthermore, the full product ion scan mode, used in LC-
ESI-triple quadrupole-MS/MS analysis, can not be employed for
the characterization of unknown DNA adducts at trace level,
because of the slow scan rate and the low sensitivity in such scan
mode. Alternatively, an analysis rely on the characteristic retention
time, but searches based on selected reaction monitoring (SRM),
do not permit de novo identification of DNA adducts.30 Taking
all this into account, the availability of a sensitive, accurate and
reliable methodology for the molecular characterization would be
of great interest. To this end, MALDI-TOF-MS, as introduced by
Karas and Hillenkamp in 1987,31 has rapidly become a valuable
technique for the analysis of a wide range of molecules with
sensitivities in the attomole range.32,33
We therefore report the development of a robust and simple
method for the detection and characterization of PAHDE-DNA
adducts based on MALDI-MS. We employ micro solid phase
extraction (µ-SPE) to remove efficiently salt and other contami-
nants of the PAHDE-DNA reaction products. Then MALDI-MS
spectra are recorded to obtain DNA-adduct mass fingerprint
(DMF). Subsequent, MS/MS-CID of selected DMF ions produce
diagnostic fragments that permit an identification and molecular
characterization of PAHDE and the nucleotide involved in the
alkylation, that is, DNA-adducts fragment fingerprint (DFF). The
usefulness of the method is afforded by characterization of adducts
formed in vitro with calf thymus DNA upon reaction with several
different PAHDE. Finally, the MALDI-MS method is also com-
pared with the 32P-postlabeling assay in regards to sensitivity
and specificity.
of fjord region PAHDE is remarkably higher than of the
,13
structurally related bay region PAHDE.7
While guanine
residues are the principal site of reaction for PAHDE derived from
planar hydrocarbons such as B[a]P,14 adenine residues are
targeted as well or more effectively than guanine residues when
the reactive metabolite is derived from a nonplanar hydrocarbon
such as B[c]Ph.15
Much research has been invested for an identification of PAH-
DNA adducts and included HPLC separation techniques coupled
with ultraviolet, or NMR, circular dichroism (CD), UV/visible,
fluorescence spectroscopy and immunoassays.16-19 However, the
most common method used for the sensitive detection of DNA
adducts is the 32P-postlabeling assay. This method involves the
reduction of genomic DNA to single deoxynucleosides-3′-
monophosphate by enzymatic hydrolysis. Then, the enzymatic
transfer of radiolabeled phosphate groups from [32P]-ATP to
nucleotides, followed by the multidimensional thin layer chro-
matography (TLC) allows the separation and detection of
modified nucleotides. This method is widely used, in part, due
to the small amounts of the DNA required and its high
sensitivity, that is, one adduct per 106-108 nonadducted
nucleotides (5 µg DNA)20,21 can be detected, but the method is
laborious with significant inter- and intralaboratory variations.22
Unfortunately, the chemical nature and molecular structure of the
alkylating agent can not be analyzed by this method and cochro-
matographic studies with DNA adduct reference materials resulted
in erroneous information as recently reported.23 There is need
for a method of high sensitivity that permits chemical identification
and characterization of the DNA adducts at the same time. In this
regard, mass spectrometry (MS) based methods were developed
for the study of PAH-DNA adducts, and are based on liquid
chromatography (LC), capillary LC or capillary electrophoresis
(CE) coupled to Electrospray ionization (ESI) tandem mass
spectrometry (MS/MS).24-26 For example, LC-ESI/MS/MS has
been used for the structural characterization of B[a]P-derived DNA
EXPERIMENTAL SECTION
Safety Considerations. Caution: B[a]PDE and B[c]ChDE are
potential mutagenic and carcinogenic agents and must be handled
with care. Protective clothing must be worn. Appropriate safety
procedures should be followed when working with this compound and
for discarded waste materials.
Chemicals and Reagents. Acetonitrile, methanol, and water
were LC-MS grade from JT Baker (Griesheim, Germany). T4
Polynucleotide kinase was from USB Corporation (Cleveland,
OH). Phosphodiesterase II from bovine spleen (SPDE), micro-
coccal nuclease from Staphylococcus aureus (MN) and all other
chemicals were of high-purity grade purchased from Sigma
Chemical Co. (St. Louis, MO).
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Med. Biol. 1994, 354, 101–12
(13) Misra, B.; Amin, S.; Hecht, S. S. Chem. Res. Toxicol. 1992, 5, 242–47
(14) Koreeda, M.; Moore, P. D.; Wislocki, P. G.; Levin, W.; Yagi, H.; Jerina,
D. M. Science 1978, 199, 778–81
(15) Dipple, A.; Pigott, M. A.; Agarwal, S. K.; Yagi, H.; Sayer, J. M.; Jerina, D. M.
Nature 1987, 327, 535–36
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J. E.; Ghei, M.; Rothman, N.; Sinha, R.; Poirier, M. C. Carcinogenesis 2002,
23, 2043–49
(18) Amin, S.; Misra, B.; Desai, D.; Huie, K.; Hecht, S. S. Carcinogenesis 1989,
10, 1971–74
(19) Lin, J. M.; Desai, D.; Chung, L.; Hecht, S. S.; Amin, S. Chem. Res. Toxicol.
1999, 12, 341–46
(20) Gupta, R. C. Cancer Res. 1985, 45, 5656–62
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1984, 5, 231–43
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R. A.; Gupta, R. C. Chem. Res. Toxicol. 2006, 19, 295–99
(24) Singh, R.; Farmer, P. B. Carcinogenesis 2006, 27, 178–96
(25) Barry, J. P.; Norwood, C.; Vouros, P. Anal. Chem. 1996, 68, 1432–38
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(27) Beland, F. A.; Churchwell, M. I.; Von Tungeln, L. S.; Chen, S.; Fu, P. P.;
Culp, S. J.; Schoket, B.; Gyorffy, E.; Minarovits, J.; Poirier, M. C.; Bowman,
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E. D.; Weston, A.; Doerge, D. R. Chem. Res. Toxicol. 2005, 18, 1306–15.
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(28) Singh, R.; Gaskell, M.; Le Pla, R. C.; Kaur, B.; Azim-Araghi, A.; Roach, J.;
Koukouves, G.; Souliotis, V. L.; Kyrtopoulos, S. A.; Farmer, P. B. Chem.
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Res. Toxicol. 2006, 19, 868–78
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Biochem. 2003, 322, 79–88
(30) Paehler, A.; Richoz, J.; Soglia, J.; Vouros, P.; Turesky, R. J. Chem. Res.
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(31) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991,
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2002, 23, 4092–103
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8574 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

Synthesis of PAH-Diol-Epoxides. The ( )-anti-7,8-dihydroxy-
9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) was syn-
thesized according to the protocol given by Yagi et al.34 The
( )-anti-9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetahydrobenzo[c]-
chrysene (B[c]ChDE) was synthesized as described by Amin
et al.9
Reactions of 2′-Deoxynucleosides and 2′-Deoxynucleo-
sides-3′-Monophosphate with PAHDE. Adducts were synthe-
sized by direct reaction of the four 2′-deoxynucleosides and the
four 2′-deoxynucleosides-3′-monophosphates (500 µg, 1 mg/mL
dissolved in water) with B[a]PDE and with B[c]ChDE (125 µg; 1
mg/mL dissolved in tetrahydrofuran) for 18 h at 37 °C in the dark.
Standard solutions were generated by purification of reaction
products as described below. The concentrations were determined
by UV spectroscopy performed on a LAMBDA 40 spectropho-
tometer (Perkin-Elmer, Waltham, MA) using known extinction
coefficients35,36
MALDI target and allowed to dry. Subsequently, the analyte
solution was deposited onto the dried matrix layer formed.
RESULTS
MALDI-MS Analysis of the B[a]PDE-2′-Deoxynucleosides
Reaction Products. The reaction mixtures of the four 2′-
deoxynucleosides and the four 2′-deoxynucleosides-3′-monophos-
phates were subjected to HLB-µ-SPE sample cleanup (see Ex-
perimental Section). Then, aliquots of the purified analytes were
loaded on MALDI target with the DHB ML sample preparation
protocol described above. The calculated and measured masses
are given in SI Table S-1, and the ion assignments are consistent
with the elemental compositions of the fragment ions depicted in
Scheme 1. A mass accuracy below 20 ppm over the whole mass
range (10-1200 m/z) was obtained and was <10 ppm for the
molecular ions ([M+H]+) at a resolution of >5000. Based on
dilution series of a 32 µM stock solution of purified B[a]PDE-
2′-deoxyguanosine-3′-monophosphate (dGp) a limit of detection
(LOD) <100 fmol loaded on target (65 pg, S/N > 3) was
determined.
Synthesis of PAHDE Modified Calf Thymus DNA and
Enzymatic Hydrolysis. The calf thymus DNA adducts of indi-
vidual PAHDE were synthesized as described by Amin et al.18
Details are given in the Supporting Information (SI).
The purified reaction products of B[a]PDE-2′-deoxyguanosine
(dG) and dGp yielded abundant protonated molecular ions
([M+H]+) at m/z 570 and m/z 650, respectively (Figure 1A, B,
and SI Figures S-1A, S-2A). Only weak cationized molecular ions
[M+Na]+ and [M+K]+ were present. The aglycon fragment
ion ([BH2]+) at m/z 454 is the result of the cleavage of the
glycosidic bond with transfer of one hydrogen from the sugar
to the base (see Scheme 1). The facile loss of the neutral
deoxyribose, is a characteristic mass-signature.26,29,39,40 The next
intense ion signal at m/z 303 corresponds to the B[a]PDE triol
([PAH]+), whereas the ions at m/z 285 and m/z 257 correspond
to a subsequent neutral losses of H2O ([PAH-H2O]+) and CO
([PAH-H2O-CO]+) from the [PAH]+ ion, all of which are
specific of the alkylating molecule. Furthermore, specific
alkylated nucleotide ions are present, that is, the 2′-deoxygua-
nosine ([dG+H]+) ion at m/z 268 and guanine ([G+H]+) ion
at m/z 152 (Figure 1A and SI Figure S-1A), whereas the ion at
m/z 348 ([dGp+H]+) is specific for the 2′-deoxyguanosine-3′-
monophosphate (Figure 1B and SI Figure S-2A). While B[a]PDE-
2′-deoxyadenosine (dA) and -2′-deoxyadenosine-3′-monophos-
phate (dAp) abundant protonated molecular ions at m/z 554 and
m/z 634 are observed (SI Figures S-1B and S-2B); whereas the
alkylated nucleotide resulted in intense signals at m/z 252 that
corresponds to [dA+H]+, and the ion at m/z 332 that corre-
sponds to [dAp+H]+. Moreover, in both spectra specific ions
for the alkylating molecule are detected together with the
adenosine [A+H]+ ion at m/z 136. The protonated molecular
ions [M+H]+ of B[a]PDE-2′-deoxycytidine (dC) and 2′-deoxy-
cytidine-3′-monophosphate (dCp) reactions yielded weaker
signals (SI Figures S-1C and S-2C) possible due to an instability
of B[a]PDE-2′-deoxycytidine adducts that undergo extensive
Microscale Solid Phase Extraction (µ-SPE) for Sample
Purification. Purification of B[a]PDE-nucleosides, -nucleotides
adduct, and DNA hydrolysates was accomplished by hydrophobic
interaction liquid chromatography using N-vinylpyrrolidone/
divinylbenzene (hydrophilic-lypophilic balance, HLB) sorbent on
self-assembled micro solid phase extraction (HLB-µ-SPE) dispos-
able columns (for details see SI). Typically, 4 pmol of B[a]PDE-
nucleosides, -nucleotides, or 12.5 ng of DNA hydrolysates were
the amount loaded onto the MALDI target. Note, representative
results of at least three independent experiments are presented
herein.
DNA-Adduct Determination by the 32P-Postlabeling As-
say. The 32P-postlabeling was carried out as described by Gupta
et al.37 with modification given by Halter et al.38 Details are
given in the SI
.
Mass Spectroscopic Analysis. MALDI-MS experiments were
performed on an Ultraflex II MALDI-TOF/TOF mass spectro-
meter equipped with a SmartBeam laser and a LIFT-MS/MS
facility. Typically, 600 spectra over a 10-1200 m/z mass range,
acquired at 100 Hz, were summed and externally calibrated using
a standard mixture composed nine peptides. (instrumentation and
software from Bruker Daltonics, Bremen, Germany). For details
see SI.
DHB Matrix Layer, MALDI Matrix Preparation Protocol.
DHB matrix layer (ML) sample preparation on AnchorChip
sample support (Bruker Daltonics) was performed as described
by Garaguso and Borlak,32 with slight modifications. The DHB
was prepared at a concentration of 5 g/L in 0.1% TFA solution
containing 30% acetonitrile and 1 g/L of ammonium phosphate
monobasic. The matrix solution, 0.5 µL, was deposited onto the
hydrolysis to B[a]P tetrols (m/z 320). In addition, the aglycon
+
is detected as weak sodiated ([BH2+Na]+) ion at m/z
(34) Yagi, H.; Thakker, D. R.; Hernandez, O.; Koreeda, M.; Jerina, D. M. J. Am.
[BH2
]
Chem. Soc. 1977, 99, 1604–11
(35) MacLeod, M. C.; Selkirk, J. K. Carcinogenesis 1982, 3, 287–92
(36) Luch, A.; Platt, K. L.; Seidel, A. Carcinogenesis 1998, 19, 639–48
(37) Gupta, R. C.; Reddy, M. V.; Randerath, K. Carcinogenesis 1982, 3, 1081–
92
(38) Halter, R.; Hansen, T.; Seidel, A.; Zieman, C.; Borlak, J. J. Occup. Environ.
Hyg. 2007, 4, 44–64
.
436 in both spectra, while an increased abundance of the B[a]P
.
.
(39) Ricicki, E. M.; Soglia, J. R.; Teitel, C.; Kane, R.; Kadlubar, F.; Vouros, P.
.
Chem. Res. Toxicol. 2005, 18, 692–99
(40) Charles, A. Barnes; Norman, H. L. Chiu Int. J. Mass Spectrom. 2009, 279,
170–75
.
.
.
Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 8575

Scheme 1. Proposed MALDI-MS/MS-CID Fragmentation Pathway of B[a]PDE-2′-Deoxynucleosides and
B[a]PDE-2′-Deoxynucleosides 3′-Phosphate. G: Guanine, A: Adenine; C: Cytosine
tetrols ions and extensive fragmentation to [PAH]+, [PAH-
H2O]+, and [PAH-H2O-CO]+ is observed in both spectra.
Presumably, differences in electrophilicity and steric constraints
of the dC and dCp compared to other deoxynucleosides may
were obtained (see SI Figure S-3C) with the characteristic ions
at m/z 112 ([C+H]+) and m/z 228 ([dC+H]+). Furthermore,
the ions at m/z 112 ([C+H]+) and m/z 308 ([dC+H]+)
characterize the dCp (SI Figure S-4C). Importantly, all spectra
yielded the ions [PAH]+ at m/z 303, [PAH-H2O]+ at m/z 285
and [PAH-H2O-CO]+ at m/z 257 that allowed the characteriza-
account for a low reaction efficiency and increased instability
,26,41
as suggested by others.11
However, no adducts were
detected in the reaction mixture with 2′-deoxythymidine (dT) and
2′-deoxythymidine-3′monophosphate (dTp).
MS/MS-CID Analysis for Characterization of B[a]PDE-
2′-Deoxynucleosides Reaction Products. The MS/MS-CID
measurements involved the molecular ions [M+H]+ at m/z 570
and m/z 650 for B[a]PDE-2′deoxyguanosine adducts (Figure
tion of the B[a]PDE alkylating molecule.
+
Aglycon Fragmentation. The MS/MS-CID of the [BH2
]
fragment (Figure 1E) resulted first in loss of H2O ([BH2-
H2O]+), a strong PAH triol fragment ion ([PAH]+) together
with the production of protonated base ion ([G+H]+). Subse-
quent isolation and MS/MS-CID fragmentation of the
[BH2-H2O]+ ion at m/z 436 (Figure 1F) show two additional
losses of H2O [BH2-2H2O]+ and [BH2-3H2O]+, the [PAH]+ ion
and its fragments to yield an abundant protonated base ion
[G+H]+ which dominated the spectrum.
PAH Triol Fragments. The MS/MS-CID of the [PAH]+ at
m/z 303 (Figure 1G) resulted in two dominant fragments at m/z
285 ([PAH-H2O]+) and m/z 257 ([PAH-H2O-CO]+). An ad-
ditional loss of water yielded the [PAH-2H2O-CO]+ ion at m/z
239.
Determination of B[a]PDE Adducts in Calf Thymus DNA
Hydrolysates by MALDI-MS. B[a]PDE was reacted with calf
thymus DNA and hydrolyzed as described in the experimental
section. One aliquot corresponding to 1.5 µg of DNA was subjected
to HLB-µ-SPE purification and about 12.5 ng of adducted DNA
were loaded onto the MALDI sample support using the DHB ML
protocol. The ion at m/z 570 corresponds to the [M+H]+ of
B[a]PDE-dG, the ion at m/z 650 corresponds to the [M+H]+
of B[a]PDE-dGp, whereas the [M+H]+ ion at m/z 554
1
C, D and SI Figures S-3A and S-4A), at m/z 554 and m/z 634 for
B[a]PDE-2′deoxyadenosine adducts, and at m/z 530 and m/z 610
for B[a]PDE-2′deoxycytidine adducts (SI Figures S-3 and S-4).
Product ions corresponding to the characteristic aglycon fragment
([BH2]+) at m/z 454 for dG and dGp, at m/z 438 for dA and
dAp, at m/z 414 for dC and dCp were readily detected. Here,
the [dN+H]+, [dNp+H]+ and [N+H]+ ions of the respective
B[a]PDE reactions are observed that allowed the characteriza-
tion of the nucleotides involved in the alkylation reaction. The
ion at m/z 268 ([dG+H]+) and at m/z 152 ([G+H]+) character-
ize the dG (Figure 1C). Likewise the ion at m/z 348 ([dGp+H]+
)
and at m/z 152 ([G+H]+) characterize the dGp (Figure 1D).
With 2′-deoxyadenosine, the fragments ions at m/z 252 ([dA+H]+
)
and m/z 136 ([A+H]+) characterize the dA (SI Figure S-3B),
whereas the ions at m/z 332 ([dAp+H]+) and m/z 136 ([A+H]+)
characterize the dAp (SI Figure S-4B). Notably, from 2′deoxy-
cytidine [M+H]+ ions, MS/MS-CID spectra of the same quality
(41) Straub, K. M.; Meehan, T.; Burlingame, A. L.; Calvin, M. Proc. Natl. Acad.
Sci. U. S. A. 1977, 74, 5285–89
.
8576 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

Figure 2. Analysis of B[a]PDE-DNA adducts by MALDI-MS and the
32P-postlabeling assay. Typical positive ion MALDI-TOF mass spec-
trum of adducts formed upon reaction of B[a]PDE with calf thymus
DNA. R, ꢀ, and γ are incomplete hydrolysis products [* ) matrix ions]
(A). Typical storage phosphor image obtained following two-
dimensional TLC; 1 indicates B[a]PDE-dG adduct; 2 indicates the
tentatively assigned B[a]PDE-dA adduct; x denotes the origin (A,
inset). Positive ion MALDI-MS/MS-CID mass spectra of the [M+H]+
ions of the DNA adducts present in DNA hydrolysate. Product ion
spectra of the B[a]PDE-dG adduct at m/z 570 (B) and the B[a]PDE-
dA adduct at m/z 554 (C) of the hydrolysates in A. The peak
annotations are detailed in Scheme 1. Positive ion MALDI-MS/MS-
CID spectra of incomplete hydrolysis products detected in the
B[a]PDE-DNA hydrolysates labeled ꢀ and R in A. Product ion spectra
obtained from the molecular ions [M+H]+: at m/z 843, identified as
B[a]PDE-dAp-dC (D); at m/z 819, identified as B[a]PDE-dCp-dC (E).
Figure 1. MALDI-TOF-MS and MS/MS-CID reference spectra of B[a]PDE
adducts. Typical positive ion MALDI-TOF mass spectra of B[a]PDE-dG (A)
and B[a]PDE-dGp (B) [* ) matrix ions]. Positive ion MALDI-MS/MS-CID
mass spectra of the molecular ions [M+H]+: at m/z 570 of the B[a]PDE-dG
in A (C), at m/z 650 of the B[a]PDE-dGp (D) in B. Positive ion MALDI-MS/
MS-CID reference spectra of specific relevant fragment ions: aglycon [BH2]+
at m/z 454 (E); [BH2-H2O]+ at m/z 436 (F) and B[a]PDE triol [PAH]+ at
m/z 303 (G) of the B[a]PDE-dG in A. The peak annotations are detailed in
Scheme 1.
characterizes B[a]PDE-dA (Figure 2A). Next to the [M+H]+
,
the aglycon ([BH2]+) at m/z 454 and at m/z 438, the protonated
deoxynucleoside ions ([dN+H]+) at m/z 268 and at m/z 252,
Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 8577

Scheme 2. Proposed MALDI-MS/MS-CID Fragmentation Pathway of B[c]ChDE-2′-Deoxynucleosides and
B[c]ChDE-2′-Deoxynucleosides 3′-Monophosphate. G: Guanine, A: Adenine; C: Cytosine
the deoxynucleoside-monophosphate ions ([dNp+H]+) at m/z
332 and at m/z 348, the protonated nucleobase fragment ions
([N+H]+) at m/z 136 and at m/z 152 of the B[a]PDE adducts
are detected. The signals of the positively charged B[a]PDE
triol fragment at m/z 303 and its fragments are also present.
Subsequently, MS/MS-CID experiments in the positive mode
are employed to confirm the identification and structural
characterization of the DNA adducts (Figure 2B, C). It should
be noted that the weak ion for the B[a]PDE-dGp could be
fragmented in one of three MS/MS-CID experiments (data not
shown). Of further interest are some ions detected at m/z 819,
m/z 843 and m/z 859 labeled as R, ꢀ, and γ, respectively (Figure
2A). Their selection and subsequent MS/MS-CID fragmentation
disclose the presence of incomplete hydrolysis products. Thus,
the MS/MS-CID of the ion at m/z 843 (Figure 2D) yield a
characteristic mass-signature allowing an identification of the
alkylated dinucleotide B[a]PDE-dAp-dC. The ions at m/z 112
([C+H]+) and at m/z 210 ([dC-H2O+H]+) indicate the presence
of dC, whereas ions at m/z 136 ([A+H]+) and m/z 332
([dAp+H]+) indicate the presence of dAp. The presence of
B[a]PDE is confirmed at m/z 303 (PAH]+) and its character-
istic fragment ions. The ion at m/z 541 is the base peak in the
spectrum and correspond to the loss of B[a]PDE (-302) from
the [M+H]+, with a subsequent loss of cytosine at m/z 430.
The ion at m/z 634 (B[a]PDE-dAp) reveal the identity of the
alkylated nucleotide, which is confirmed by the presence of
the [BH2]+ ion at m/z 438 and by the ion at m/z 732 resulting
from the loss of a cytosine (-111) of the [M+H]+. Moreover,
the absence of a B[a]PDE-dC aglycon ion [BH2]+ at m/z 414
and a B[a]PDE-dCp ion at m/z 610 defines the site of adduction
of the dAp. Likewise, MS/MS-CID spectra are obtained from
the ion at m/z 819 leading to unambiguous identification and
characterization of the dinucleotide B[a]PDE-dCp-dC (Figure
2
E). An intense ion at m/z 112 ([C+H]+) and at m/z 308
([dCp+H]+) define the presence of deoxycytidine. The ions
at m/z 610 (B[a]PDE-dCp) and the aglycon ion at m/z 414
characterize the alkylation event, whereas the ion at m/z 708
represents the loss of a cytosine (-111) from the [M+H]+.
The ion at m/z 517 corresponds to the loss of B[a]PDE (-302)
from the [M+H]+ and a subsequent loss of cytosine at m/z
406, are further informative ions in this spectrum. The MS/
MS-CID of the weak ion at m/z 859 produced poor spectra.
Determination of B[a]PDE Adducts in Calf Thymus DNA
by the 32P-Postlabeling Assay. Ten µg of B[a]PDE-DNA
hydrolysates that were also used for MALDI-MS studies were
enriched by butanol extraction. Eventually, an equivalent of 2 ng
of 32P-Postlabeled DNA was applied to multidimensional TLC
(Figure 2A inset). Here, one intense spot corresponding to
B[a]PDE-deoxyguanosine and a faint spot tentatively assigned to
the B[a]PDE-deoxyadenosine adduct, were detected, giving rise
to a relative adduct level (RAL) of ∼25 adducts per 106 deoxy-
nucleotides and ∼2 adducts per 106 deoxynucleotides for
B[a]PDE-deoxyguanosine and B[a]PDE-deoxyadenosine, re-
spectively.
Determination of B[c]ChDE Adducts in Calf Thymus DNA
Hydrolysates by MALDI-MS. To further demonstrate the reli-
ability of the MALDI-MS based methodology, DNA adducts
derived from reaction with ( )-anti-benzo[c]chrysene-9,10-diol-
11,12-epoxide (B[c]ChDE), a metabolic activation product of
benzo[c]chrysene, were studied. In Scheme 2 the major predicted
fragment and the fragmentation pathways of B[c]ChDE-DNA
adduct are depicted. Thus, one aliquot of hydrolysate that
8578 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

corresponds to 1.5 µg of DNA was subjected to HLB-µ-SPE and
a volume corresponding to 12.5 ng of modified DNA was loaded
onto the MALDI sample support. The ion at m/z 596 corresponds
to the [M+H]+ of B[c]ChDE-dG, the ion at m/z 580 is the
[M+H]+ of B[c]ChDE-dA and the ion at m/z 556 corresponds
to the [M+H]+ of B[c]ChDE-dC, all of which were readily
detected (Figure 3A). In addition, MALDI-TOF analysis yielded
signals of the 2′-deoxynucleoside-3′-monophosphate adducts: the
B[c]ChDE-dGp at m/z 676, the B[c]ChDE-dAp at m/z 660 and
the B[c]ChDE-dCp at m/z 636. Next to the [M+H]+, unique
mass-signature ions for the B[c]ChDE and the alkylated
nucleosides were also detected. Here, the characteristic [BH2]+
ions at m/z 480 for B[c]ChDE-dG, at m/z 464 for B[c]ChDE-
dA and at m/z 440 for B[c]ChDE-dC are noted. The protonated
deoxynucleoside ions ([dN+H]+), at m/z 268 and at m/z 252,
the deoxynucleoside-monophosphate fragment ([dNp+H]+) at
m/z 332 and 348 and the protonated nucleobase fragment
([N+H]+) at m/z 112, 136, and 152 are also detected. For the
B[c]ChDE triol ([PAH]+) at m/z 329 mainly loss of H2O, the
[PAH-H2O]+ ion at m/z 311 are observed. Subsequently, MS/
MS-CID experiments in the positive mode were carried out to
obtaine fragmentation spectra of B[c]ChDE-dG, B[c]ChDE-
dA, B[c]ChDE-dC (Figure 3B-D), B[c]ChDE-dGp and of B[c]-
ChDE-dAp (Figure 3E and F). Typically, the B[c]ChDE triol
[PAH]+ yielded a weak ion at m/z 329, an intense fragment
ion [PAH-H2O]+ at m/z 311 as well as [PAH-2H2O]+ at m/z
293. Moreover, consecutive loss of CO and water from the ion
at m/z 311 produce the ions [PAH-H2O-CO]+ at m/z 283 and
[PAH-2H2O-CO]+ at m/z 265 (Figure 3B inset and Scheme 2).
This characteristic fragmentation pattern could be further evi-
denced in MS/MS-CID esperiments and together with the
[M+H]+ and the aglycon [BH2]+ all the structural characteristic
specific ions are present in each spectrum in agreement with
the elemental compositions of the fragment ions depicted in
Scheme 2. An identification and molecular characterization of the
DNA adducts could thus be definitively confirmed. Moreover, the
MS/MS-CID spectrum of B[c]ChDE-dC [M+H]+ ion at m/z 556
yield, next to its specific fragments, additional ions at m/z 136
and at m/z 480 that correspond with high probability to the
[N+H]+ of dA and the aglycon ion [BH22]+ of B[c]ChDE-dG,
respectively. We propose that the isobaric fragment at m/z
556.1478 derived from a dinucleotide (see below) could not
be efficiently discriminated by the ion-selector in the fragmen-
tation process. We additionally observed ions at m/z 845, m/z
869, m/z 884 and m/z 893, which are labeled R, ꢀ, γ, and δ in
Figure 3A, respectively. The molecular ion at m/z 893 allowed
an identification of the B[c]ChDE-dAp-dA dinucleotide (Figure
4A). The ion at m/z 660 (B[c]ChDE-dAp) reveal the identity of
the alkylated nucleotide, which is confirmed by the presence of
the aglycon [BH2]+ at m/z 464 and by the ion at m/z 758 derived
from the loss of adenine (-135) of the [M+H]+. The CID
spectrum yielded the specific ions for the deoxyadenosine: the
ions at m/z 136 ([A+H]+), at m/z 252 ([dA+H]+) and m/z
332 ([dAp+H]+), whereas the ion at m/z 565 corresponded to
the loss of B[c]ChDE (-328) from the [M+H]+. The selection
and subsequent MS/MS-CID fragmentation of the [M+H]+ ion
at m/z 869 led to an identification of the dinucleotide
Figure 3. Analysis of B[c]ChDE-DNA adducts by MALDI-MS and the 32P-
postlabeling assay. Typical positive ion MALDI-TOF mass spectrum of
adducts formed upon reaction of B[c]ChDE with calf thymus DNA. R, ꢀ, γ,
and δ are incomplete hydrolysis products [* ) matrix ions] (A). Typical
storage phosphor image obtained following two-dimensional TLC; 1 indi-
cates B[c]ChDE-dG adduct; 2 indicates B[c]ChDE-dA adduct; 3 indicates
the tentatively assigned B[a]PDE-dC adduct; x denotes the origin (A, inset).
Positive ion MALDI-MS/MS-CID mass spectra of the [M+H]+ ions of DNA
adducts present in DNA hydrolysate. Product ion spectra of the B[c]ChDE-
dG adduct at m/z 596 (B), the B[c]ChDE-dA adduct at m/z 580 (C), the
B[c]ChDE-dC adduct at m/z 556 (D), the B[c]ChDE-dGp at m/z 676 (E)
and the B[c]ChDE-dAp adduct at m/z 660 (F) of the hydrolysates in A.
Expanded region of B for details of the B[c]ChDE triol ion [PAH]+
characteristic mass-signature (B, inset). The peak annotations are detailed
in Scheme 2.
Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 8579

be due to the loss of thymine (-126) from the [M+H]+ (if
B[c]ChDE-dAp-dT). Noteworthy, the ion at m/z 556 is isobaric
to the B[c]ChDE-dC (at m/z 556.2078) but could correspond
to the loss of B[c]ChDE (-328) from the [M+H]+. However,
selection and fragmentation lead to the spectrum depicted in
Figure 3D.
Determination of B[c]ChDE Adducts in Calf Thymus DNA
by the 32P-Postlabeling Assay. Ten µg of B[c]ChDE-DNA
hydrolysates that was also used for MALDI-MS analysis were
enriched by butanol extraction followed by 32P-postlabeling with
2 ng of adducted DNA hydrolysate being applied to multidi-
mensional TLC (see inset in Figure 3A). Here, two radioactive
intense spots corresponding to B[c]PhDE-deoxyguanosine and
B[c]PhDE-deoxyadenosine adducts and a third very faint spot
tentatively assigned as B[c]PhDE-deoxycytidine adduct were
detected. The RAL was determined as ∼59 adduct per 106
deoxynucleotides for B[c]PhDE-deoxyguanosine, ∼18 adduct
per 106 deoxynucleotides for B[c]PhDE-deoxyadenosine and
∼3 adduct per 107 deoxynucleotides for B[c]PhDE-deoxycyti-
dine.
DISCUSSION
This study aimed for the development of a rapid and sensitive
method for the detection, identification, and characterization of
PAHDE-DNA adducts by MALDI-TOF-MS and the following
experimental conditions need to be considered. First, to facilitate
the acquisition of MALDI-MS and MS/MS-CID mass spectra, the
matrix sample preparation should produce consistent, reproduc-
ible, and long-lasting analyte signals. This is achieved by the
homogeneous distribution of the matrix-analyte crystals on the
sample support. Second, the matrix should produce a limited
number of identifiable matrix peaks, and should not be affected
by the presence of salts, buffers, and other common sample
components, which could result in matrix clusters formation and
ion suppression. Third, the matrix influences the extent of
desorption/ionization-induced fragmentation of analytes.42 No-
table, the 3-hydroxypicolinc acid (HPA) matrix proved useful for
the analysis of oligonucleotides,43 whereas R-cyano-4-hydroxycin-
namic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) worked
well for the analysis of proteins and peptides.32,44 For an analysis
of PAHDE-DNA adducts these matrices exhibited different
characteristics with respect to appearance of the crystals, the
production of matrix ions, signal reproducibility, and the degree
of analyte fragmentation (data not shown). We found the DHB
prepared by the matrix layer (ML) method32 to be the matrix of
choice for an analysis of PAHDE and PAHDE-DNA adducts. An
improved homogeneity of the matrix crystals, enhanced sensitivity
and reduced cationized matrix and analyte molecular ions by the
addition of ammonium phosphate as matrix “dopant” was ob-
served. Furthermore, the DHB ML sample preparation delivered
consistent signals for hundreds of laser shots at one location to
facilitate the acquisition of MALDI-MS and MS/MS-CID spectra.
Likewise, the MALDI signals are long-lived, and a single sample
Figure 4. MALDI-MS/MS-CID spectra of incomplete hydrolysis
products detected in of B[c]ChDE-DNA hydrolysates. Positive ion
MALDI-MS/MS-CID mass spectra of the dinucleotides [M+H]+ present
the DNA hydrolysates labeled δ, γ, ꢀ and R in Figure 3A. The [M+H]+
ions: at m/z 893, identified as B[c]ChDE-dAp-dA (A); at m/z 884 (B);
at m/z 869, identified as B[c]ChDE-dAp-dC (C); at m/z 845, identified
as B[c]ChDE-dCp-dC (D).
B[c]ChDE-dAp-dC (Figure 4C). Thus, ions at m/z 112 ([C+H]+
)
and at m/z 210 ([dC-H2O+H]+) identify the dC, whereas ions
at m/z 136 ([A+H]+) and 332 ([dAp+H]+) identify the dAp as
the nucleotide components. The specific ion at m/z 660 (B[c]-
ChDE-dAp) reveal the identify of the alkylated nucleotide which
is confirmed by the [BH2]+ ion at m/z 464 and by the ion at
m/z 758 which resulted from the loss of cytosine (-111) of
the [M+H]+. Moreover, the ion at m/z 541 corresponded to
the loss of B[c]ChDE (-328) from the [M+H]+ with subse-
quent loss of cytosine (-111) at m/z 430. The MS/MS-CID
spectrum of the ion at m/z 845 identified the dinucleotide
B[c]ChDE-dCp-dC (Figure 4D).The ion at m/z 636 (B[c]ChDE-
dCp) revealed the identity of the alkylated nucleotide which is
confirmed by the presence of the [BH2]+ at m/z 440 and by the
ion at m/z 734 derived from loss of cytosine (-111) of the
[M+H]+. Further specific ion for the nucleotide components
includes the ions at m/z 112 ([C+H]+) and a weak [dCp+H]+
ion at m/z 308. Regarding the ion at m/z 884 (Figure 4B) the
incomplete fragmentation pattern of the CID spectrum leads us
to tentatively assigne fragment ions. Thus, the ion at m/z 660,
the ion at m/z 464 (as its aglycon [BH2]+) together with ions at
m/z 136 ([A+H]+) and m/z 332 ([dAp+H]+) define the
presence of B[c]ChDE-dAp, whereas the ion at m/z 758 could
(42) Stemmler, E. A.; Hettich, R. L.; Hurst, G. B.; Buchanan, M. V. Rapid
Commun. Mass Spectrom. 1993, 7, 828–36
(43) Wu, K. J.; Steding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993,
7, 142–46
(44) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 432–
35
.
.
.
8580 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010

can easily be used for extensive MS/MS-CID experiments. In
addition, the purity of the sample is of great importance with DNA
hydrolysates containing huge amounts of unreacted nucleotides,
buffers, and enzymes which could hinder the matrix crystallization,
the reproducibility of the spectra, and provoke ion suppression
effects to impair the MALDI-MS analysis. Samples were therefore
subjected to solid phase extraction for purification/enrichment
that either consisted of silica-based C18, polymer-based styrene/
divinylbenzene (SDB) or N-vinylpyrrolidone/divinylbenzene (hy-
drophilic-lypophilic balance, HLB) bed materials. While many
reports described the use of one of these resins alone or in
combination for the purification/enrichment of PAHDE-DNA
adducts,25,26,29 the HLB copolymer sorbent proofed valuable
enabling increased recovery and enhanced loading capacity as
compared to other resins. Therefore, we implemented the HLB
sorbent and developed self-assembled disposable micro columns
(HLB-µ-SPE, see Experimental Section) to meet the sample
amount and the volume used for MALDI-MS sample preparation.
As shown in SI Table S-1 the reaction products of B[a]PDE
with the four 2′-deoxynucleosides and the four 2′-deoxynucleo-
sides-3′-monophosphates purified by HLB-µ-SPE and were mea-
sured with a mass accuracy of <10 ppm at a resolution of >5000,
and a sensitivity of <100 fmol loaded on target.
diagnostic fragment ions which enabled improved characteriza-
tion. Indeed, fragmentation of the protonated adducted base
([BH2]+) produced [G+H]+ and involves hydrogen transfer
from PAH triol. This, would explain the positive charged
[PAH]+ fragment. Moreover, the fragmentation of the [PAH]+
produced the fragments [PAH-H2O]+, [PAH-H2O-CO]+ and
[PAH-2H2O-CO]+ which are specific and characteristic of the
PAH triol moiety. All together these information allow the
identity of the PAH and the number and type of ring substituent
to be defined. In fact, these diagnostic fragments were used
to structurally differentiate diastereoisomers of PAH and PAH-
DNA adducts.42,47,48 Therefore, both the DMF and DFF permitted
an identification and characterization of B[a]PDE adducts formed
with the four 2′-deoxynucleosides and the four 2′-deoxynucleosides
3′-monophosphates (see Figure 1 and SI Figures S-1-S-4).
Consequently, we applied this methodology for analysis of
adducts formed with calf thymus DNA upon reaction with
B[a]PDE. Based on the full MALDI-TOF mass spectrum, a simple
comparison with the reference DMF allowed specification of the
molecular ion and characteristic fragment ions of the B[a]PDE-
DNA adducts. Readily molecular ions and characteristic fragment
for deoxynucleotides and deoxynucleosides were detected. The
B[a]PDE-dG at m/z 570, B[a]PDE-dGp at m/z 650 and B[a]PDE-
dA at m/z 554, B[a]PDE-dCp at m/z 610 DNA adducts were the
main adducted molecules detected with calf thymus DNA.
Notably, the latter adduct could be identified as a dinucleotide
component. Some of the commercially available SPDEs, used in
the DNA hydrolysis, contain 3′-phosphatase activity.49 This may
explain why the B[a]PDE-DNA adduct hydrolysates contained
both the alkylated nucleotides and nucleosides. We exclude the
possibility that these PAHDE-2′-deoxynucleosides are fragmenta-
tion products from the equivalent 3′-monophosphate as we never
observed such a fragmentation in MS or MS/MS-CID reference
spectra. Further adducts were identified as dinucleotide resulting
from incomplete DNA hydrolysis. That is, the ions corresponding
to the B[a]PDE-dCp-dC and the B[a]PDE-dAp-dC dinucleotides.
Moreover, the MALDI-MS/MS-CID analysis allowed the specifica-
tion and characterization of the alkylation event as the B[a]PDE-
dCp and B[a]PDE-dAp adducts, respectively. In one report and
next to B[a]PDE-dGp ions at higher m/z were observed. However,
the investigators were unable to determinate the identity and to
characterize further adducts, despite the elaborate SPE enrich-
ment prior to CE-coupled ESI-MS.25 Incomplete DNA hydrolysis
products are probably formed because the alkylated nucleotides
are not efficiently recognized from MN and/or SPDE due to
sterically unfavorable interactions of their modified structure.
Nonetheless, the efficiency of MN/SPDE enzymatic hydrolysis
could be improved by a preceding DNA hydrolysis step with DNA-
ase I or benzonase endonucleases which release 5′-phosphate
oligonucleotides. Importantly, the digestion protocol used in this
study is widely used for 32P-postlabeling assay.
The molecular ions ([M+H]+) corresponded to the monoalky-
lated B[a]PDE adducts of dG, [M+H]+ at m/z 570; dGp,
[M+H]+ at m/z 650; dA, [M+H]+ at m/z 554; dAp, [M+H]+
at m/z 634; dC, [M+H]+ at m/z 530; dCp, [M+H]+ at m/z
610. In the reaction mixture with 2′-deoxythymidine (dT) and
2′-deoxythymidine-3′monophosphate (dTp), no adducts were
detected presumably due to low alkylation efficiency. This is
consistent with previous reports using similar condition but
other ionization techniques.26 Noteworthy, cationazed
([M+Na]+ or [M+K]+) molecular ions were weak or absent,
which is a particular advantage for the interpretation of spectra.
The MALDI-TOF spectra of PAHDE-DNA adducts together
with the molecular ion ([M+H]+) provide typical mass-
signature specific for the alkylating molecule and the nucleotide
involved in the alkylation reaction. Indeed, next to the [M+H]+,
the aglycon fragment ([BH2]+), the protonated 2′-deoxynucleo-
side ([dN+H]+) or the 2′-deoxynucleoside-3′-monophosphate
fragments ([dNp+H]+), the protonated nucleobase ([N+H]+)
together with signals of the positively charged B[a]PDE triol
fragment ([PAH]+) are the observed mass-signature (Figure
1
A, B and SI Figures S-1 and S-2). Such fingerprints can thus be
defined as DNA-adduct mass fingerprint (DMF). Based on the
DMF, the [M+H]+ and specific ions were selected for MS/
MS-CID measurements to obtain definitive structural diagnostic
fragment ions (Figure 1 C, D and SI Figures S-3 and S-4), that
is, DNA-adducts fragment fingerprint (DFF). In Scheme 1 the
major fragment and the fragmentation pathways are depicted.
Several different investigations reported the detection of the
aglycon ion [BH2]+, as major ion product for the characterization
of DNA adducts.28,29,39,40,45,46 The new developed method allowed
further fragmentation of this and other DFF ions component on
the same target spot. Thus, MS/MS-CID fragmentation of the
[BH2]+ and the [PAH]+ ions were achieved leading to additional
Noteworthy, cationazed ([M+Na]+ or [M+K]+) molecular
ions were weak or absent. Thus sufficient cleanup of analytes
(47) Chiarelli, M. P.; Chang, H. F.; Olsen, K. W.; Barbacci, D.; Huffer, D. M.;
Cho, B. P. Chem. Res. Toxicol. 2003, 16, 1236–41
(48) Huffer, D. M.; Chang, H. F.; Cho, B. P.; Zhang, L. K.; Chiarelli, M. P. J. Am.
Soc. Mass Spectrom. 2001, 12, 376–80
.
.
(45) Annan, R. S.; Giese, R. W.; Vouros, P. Anal. Biochem. 1990, 191, 86–95
(46) Lay, J. O., Jr.; Chiarelli, M. P.; Bryant, M. S.; Nelson, R. W. Environ. Health
Perspect. 1993, 99, 191–93
.
(49) Suzuki, N.; Prabhu, P. M.; Shibutani, S. In Optimization in Drug Discovery,
Chapter 17; Zhengyin, Y., Caldwell, G. W., Ed.; Humana Press: Totowa,
NJ, 2004.
.
Analytical Chemistry, Vol. 82, No. 20, October 15, 2010 8581

was achieved. In previous report using standard solutions of
adducts the cationazed form ions were the base peaks in the
spectra rendering an interpretation challenging. In contrast the
here reported HLB-µ-SPE purification/enrichment protocol is
simple and robust.
CONCLUSIONS
A rapid MALDI-TOF-MS based method has been developed,
and applied for the sensitive detection, identification and charac-
terization of prototypic PAH-DNA adducts derived from B[a]PDE
and B[c]ChDE that were reacted with calf thymus DNA. We
evidence that MALDI-TOF and MS/MS-CID spectra yielded
characteristic fragmentation patterns that allow identification of
deoxyguanosine, deoxyadenosine, and deoxycytidine DNA ad-
ducts. The spectra are simple to interpret and there is a notable
absence of cationized sample and matrix ions. Overall the method
is sensitive, and nanogramms of hydrolyzed DNA are sufficient
for the identification and characterization of the adducts. When
compared with the 32P-postlabeling assay a distinct advantage
of the method is the unambiguous identification of DNA
adducts and the possibility to identify the chemical nature of
the alkylating agent. Moreover, our method allowed the
simultaneous and unambiguous detection and identification of
deoxynucleotide and deoxynucleosides PAHDE adducts at the
same time. In addition, the analysis time is drastically reduced
and a dedicated radioactivity laboratory is not required. In the
long term, a database of DNA-adducts mass fingerprints and
fragmentation pattern of reactions products of several different
PAH with nucleotides and nucleoside will be developed for an
automated detection of DNA-adducts.
We also compare findings obtained by MALDI-MS with the
32P-postlabeling assay. Here, two radioactive spot were detected
corresponding to B[a]PDE-deoxyguanosine and the B[a]PDE-
deoxyadenosine adducts. Importantly, dinucleotides which are
also substrate of the T4 polynucleotide kinase were not
detected. Hence, a comparison of the two methodologies clearly
demonstrates that only MALDI-MS provided molecular speci-
fication to characterize the nucleotide and the alkylating agent
involved. Additionally, the B[a]PDE-dCp adduct was identified
by MALDI-MS. Thus, MALDI-MS provided comprehensive
information such as the detection and identification of deoxy-
nucleotides, deoxynicleosides and dinucleotides adducts, that
was not detected by the 32P-postlabeling assay.
The reliability of our MALDI-MS based methodology is further
demonstrated by the detection, identification, and characterization
of the adducts derived from reaction of calf thymus DNA with
( )-anti-benzo[c]chrysene-9,10-diol-11,12-epoxide (B[c]ChDE). The
B[c]ChDE, is an intriguing PAH since is characterized by the
presence of a bay and a fjord region within the same molecule
(see Scheme 2). The location of the epoxide in the sterically
hindered fjord region determines its high carcinogenic activity.9
With the 32P-postlabeling assay only three radioactive spot were
detected corresponding to B[c]ChDE-deoxyguanosine, -deoxy-
adenosine, and deoxycytidine adducts. Applying the newly
developed method we were able to readily identify a total of
six B[c]ChDE-DNA adduct as 2′-nucleosides and 2′-nucleo-
sides-3′monophosphate: the B[c]ChDE-dG at m/z 596, the
B[c]ChDE-dGp at m/z 676, the B[c]ChDE-dA at m/z 580, the
B[c]ChDE-dAp at m/z 660, the B[c]ChDE-dC at m/z 556 and
the B[c]ChDE-dCp at m/z 636. In addition, three dinucleotides
adducts were also detected: the B[c]ChDE-dAp-dA, the B[c]-
ChDE-dAp-dC, and the B[c]ChDE-dCp-dC. MS/MS-CID frag-
mentation confirmed the identification and allowed the molec-
ular characterization of the nucleotides and nucleosides with
B[c]ChDE as the alkylating molecule. Again, a comparison of
the two methodologies confirm that only MALDI-MS led to a
comprehensive analysis and provided molecular specification
for an identification and characterization of the adducts formed.
In regard to the sensitivity, B[a]PDE-deoxycytidine was de-
tected by the 32P-postlabeling with a RAL of ∼3 adduct per 107
deoxynucleotides. Consequently, the MALDI-TOF-MS method
has at list a similar sensitivity.
ACKNOWLEDGMENT
We wish to thank Ines Voepel, Ute Sanger, Doreen Schellbach
and Jyh-Ming Lin for the technical help. We thank also Dott. Maria
Stella Ritorto for the constructive discussion.
ABBREVIATIONS
PAH
polycyclic aromatic hydrocarbons
B[a]PDE ( )-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide
B[c]ChDE( )-anti-benzo[c]chrysene-9,10-diol-11,12-epoxide
DHB
ML
2,5-dihydroxybenzoic acid
matrix layer
SUPPORTING INFORMATION AVAILABLE
Experimental procedures, spectral data and images. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Received for review June 25, 2010. Accepted September
7, 2010.
AC101568H
8582 Analytical Chemistry, Vol. 82, No. 20, October 15, 2010