UV-Induced Bond Modifications in Thymine and Thymine

Jun 25, 2010 - Bradley B. Schneider , Erkinjon G. Nazarov , Thomas R. Covey ... Helko Borsdorf , Thomas Mayer , Mashaalah Zarejousheghani , Gary A...
0 downloads 0 Views 275KB Size
Download PDF
Anal. Chem. 2010, 82, 6163–6167

UV-Induced Bond Modifications in Thymine and Thymine Dideoxynucleotide: Structural Elucidation of Isomers by Differential Mobility Mass Spectrometry Antony St-Jacques,† Janna Anichina,† Bradley B. Schneider,‡ Thomas R. Covey,‡ and Diethard K. Bohme*,† Department of Chemistry, Centre for Research in Mass Spectrometry, York University, Toronto, ON, Canada M3J 1P3, and AB SCIEX Concord, ON, Canada L4K 4V8 Differential mobility spectrometry has been applied to reveal the occurrence of isomerization of thymine nucleobase and of thymine dideoxynucleotide d(5′-TT-3′) due to bond redisposition induced by UV irradiation at 254 nm of frozen aqueous solutions of these molecules. Collision-induced dissociation (CID) spectra of electrosprayed photoproducts of the thymine solution suggest the presence of two isomers (the so-called cyclobutane and 6,4-photoproducts) in addition to the proton-bound thymine dimer, and these were separated using differential mobility spectrometry/mass spectrometry (DMS/ MS) techniques with water as the modifier. Similar experiments with d(5′-TT-3′) revealed the formation of a new isomer of deprotonated thymine dideoxynucleotide upon UV irradiation that was easily distinguished using DMS/MS with isopropanol as the modifier. The results reinforce the usefulness of DMS/MS in isomer separation. Simulations of UV-induced DNA damage with thymine and other related biomolecules have been hampered by the identification of isomeric photoproducts. Early UV irradiation experiments with frozen solutions of thymine suggested formation of a covalently bound photoproduct between two thymine nucleobases with the structure of the cyclobutane dimer1 (see Scheme 1) that was confirmed later by crystallography of the sodium salt of the thymine dimer.2 Formation of the dimer was shown to be a reversible process3 with an equilibrium position between thymine and its dimer that was dependent on the wavelength of the UV irradiation. At longer wavelengths, the equilibrium is shifted toward the dimer (λ > 280 nm), whereas at a shorter wavelength (λ < 240 nm) the monomer predominates.4 Furthermore, X-ray diffraction experiments suggested the formation of a different covalently bound dimer in a frozen solution of thymine (T) * To whom correspondence should be addressed. E-mail: dkbohme@ yorku.ca. † York University. ‡ AB SCIEX Concord. (1) Rorsch, A.; Beukers, R.; IJlstra, J.; Berends, W. Rec. Trav. Chim. 1958, 77, 423–429. (2) Wei, C. H.; Einstein., J. R. Acta Crystallogr. 1984, B40, 271–279. (3) Setlow, J. K.; Setlow., R. B. Nature 1963, 197, 560–562. (4) Setlow, R. B.; Setlow., J. K. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 1250– 1257. 10.1021/ac100977b  2010 American Chemical Society Published on Web 06/25/2010

Scheme 1. Structures of the Cyclobutane Photoproduct (Top Left), the 6,4-Photoproduct (Top Right) and the Proton-Bound Dimer (Bottom)

exposed to UV irradiation (λ ) 260 nm).5 The structure of this species, the so-called 6,4-photoproduct, also is presented in Scheme 1.5 The formation of thymine dimers in isolated DNA that was demonstrated in a number of studies6,7 is consistent with results obtained for the frozen solutions of thymine nucleobase. (However, no freezing of DNA samples was performed.) The necessity to freeze thymine solutions to obtain covalently bonded photoproducts together with the observed formation of covalent bonds between two adjacent thymines of the same strand in irradiated DNA at room temperature gave rise to a hypothesis that proposed a steric requirement for inter- or intramolecular covalent bond formation between adjacent thymines.8,9 In order to probe further the formation of multiple structural isomers, we investigated the photoproducts of UV-induced polymerization of thymine nucleobase and thymine dideoxynucleotide d(5′-TT-3′) using electrospray ionization (ESI) and differential mobility spectrometry/mass spectrometry (DMS/MS) which allows the separation of structural isomers of ions based on differences in velocity when drifting through high versus low Karle, I. L.; Shih Yi Wang; Varghese, A. J. Science 1969, 164, 183–184. Beukers, R.; Ijlstra, J.; Berendes, W. Rec. Trav. Chim. 1960, 79, 101. Setlow, R. B.; Carrier, W. L. Proc. Natl. Acad. Sci. 1964, 51, 226–231. Beukers, R.; Eker, A. P. M.; Lohman, P. H. M. DNA Repair 2008, 530– 543. (9) Douki, T.; Court, M.; Cadet, J. J. Photochem. Photobiol. B: Biol. 2000, 54, 145–154. (5) (6) (7) (8)

Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

6163

electric fields.10-12 A “modifier” molecule was added in attempts to further improve isomeric resolution since the addition of a neutral molecule (usually the vapor of a volatile liquid) to the DMS cell recently has been found to significantly influence the resolution and the capacity of the peaks in DMS spectra.13 EXPERIMENTAL SECTION Collision-induced dissociation (CID) experiments were performed with an API 2000 ESI triple quadrupole mass spectrometer (AB SCIEX, Concord, Ontario) to explore the nature of the dissociation of the sprayed ions. The CID experiments provide unique fragment ions to identify these isomers. Differential mobility spectrometry/mass spectrometry (DMS/ MS) was employed to separate the isomeric electrosprayed products of photopolymerization. For the differential mobility mass spectrometry measurements, a planar DMS was interfaced to an AB SCIEX 5500 QTRAPTM instrument equipped with a standard TurboVTM electrospray ionization source. Fundamentals of differential mobility mass spectrometry along with the details of the instruments have been described previously.14 The dimensions of the DMS cell were 30 mm long × 10 mm wide with a 1 mm gap between the electrodes in order to provide high transmission efficiency with the gas flow provided by the mass spectrometer. The DMS cell was sealed to the mass spectrometer inlet orifice and enclosed within a curtain chamber as previously described.14 A constant flow of 3.3 L min-1 of nitrogen “curtain gas” was provided to the curtain chamber, providing both a gas outflow from the curtain plate (≈ 0.5 L min-1) and the transport gas flow through the DMS cell and into the vacuum system of the mass spectrometer (≈ 2.8 L min-1). The curtain gas temperature was maintained at ≈100 °C. We chose water and isopropanol with v/v concentrations of about 5% and 1.5% of the curtain gas, respectively, as modifiers in the mobility-controlled separation experiments reported here. An asymmetric RF field was generated using a bisinusoidal waveform from a custom tuned-harmonic generator.14 The generator was operated at 3 MHz, providing separation voltage (SV) with peak magnitude from 0 to 3333 V (5000 V peak-to-peak). The direct current compensation voltage (CV) could be scanned at ±100 V or set to a particular value within that range. In this study, we varied the CV from -25 V to +25 V. When the parent ion leaving the DMS is monitored by Q1 as a function of CV (from -100 V to +100 V), a mobility spectrum is obtained that separates the various isobaric isomers. Q3 is then set to the masses of the characteristic fragment ions of each isomer in the multiple reaction monitoring (MRM) mode, and the CV is scanned as before. The DMS resolution was controlled by metering gas (the “throttle gas”) into a juncture chamber located between the DMS outlet (10) Eiceman, G.; Karpas. Z. In Ion Mobility Spectrometry, 2nd ed.; CRC Press, Taylor and Francis LLC: Boca Raton, FL, 2005. (11) Krylov, E. V.; Nazarov, E. G.; Miller, R. A. Int. J. Mass Spectrom. 2007, 76, 226–230. (12) Shvartsburg, A. Differential Ion Mobility: Non-Linear Ion Transport and Fundamentals of FAIMS; CRC Press, Taylor and Francis LLC: Boca Raton, FL, 2008. (13) Eiceman, G. A.; Krylov, E.; Krylova, N.; Nazarov, E. G.; Miller, R. A. Anal. Chem. 2004, 76, 4937–4939. (14) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Eur. J. Mass Spectrom. 2010, 16, 57–71.

6164

Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

Figure 1. Collision-induced dissociation spectra of m/z 253 isolated from an aqueous solution of thymine before (top) and after (bottom) irradiation of the frozen solution with UV light at 254 nm. The spectra were acquired in the positive ion mode and averaged for the range of the laboratory collision voltage from 1 to 30 V.

and mass spectrometer inlet orifice as previously described.15 The resolution settings were low, medium, and high, corresponding to throttle gas flows of 0, 1, and 1.5 L min-1, respectively. The thymine nucleobase (Sigma, 99% min.) and thymine dideoxynucleotide d(5′-TT-3′) (ACGT corporation, Toronto, ON) were obtained commercially. Dideoxynucleotide was desalted and cartridge purified. Thawed aqueous solutions of thymine nucleobase and of thymine dideoxynucleotide d(5′-TT-3′) were electrosprayed before and after exposing frozen solutions to UV light at the wavelength of 254 nm. We focused on the protonated dimer ion of thymine and the deprotonated thymine dideoxynucleotide that were the major ions observed in the mass spectrum of the spray. Collision-induced dissociation (CID) experiments were performed first to explore the nature of the dissociation of these two sprayed ions before and after irradiation. DMS/MS was employed subsequently to separate any possible isomers with water or isopropanol chosen as the modifier. The mobilities of the species were studied by monitoring selected MRM transitions for the ions. RESULTS AND DISCUSSION CID Experiments with Dimers of Thymine Nucleobase. The positive-ion mass spectrum of the electrosprayed water solution of the thymine nucleobase before irradiation revealed the presence of a protonated thymine-thymine (T-T) dimer at m/z 253 (see Figure 1). The MS/MS spectrum of this ion suggests that this dimer is proton bound since its dissociation proceeds via the loss of a mass corresponding to the neutral nucleobase to form a protonated monomer at m/z 127. MS/MS measurements performed after exposure of the frozen aqueous samples of thymine to UV irradiation at 254 nm for 1 h revealed two new primary dissociation pathways for the ion at m/z 253: the loss of a water molecule to produce m/z 235 and a neutral loss of 43 that (15) Schneider, B. B.; Covey, T. R.; Coy, S. L.; Krylov, E. V.; Nazarov, E. G. Int. J. Mass Spectrom. 2010, DOI: 10.1016/j.ijms.2010.01.006, in press.

Figure 2. Negative ion mode MS/MS spectra of the m/z 545 measured for aqueous solutions of d(5′-TT-3′) before (top) and after (bottom) irradiation with UV light. The spectra were averaged over a range of the laboratory collision voltage from -30 to -1 V.

can correspond to the loss of C2H5N or HCNO. The additional dissociation pathways that were observed suggest the formation of one or more new structural isomers of the protonated dimer in the solution of the irradiated thymine. Several isomeric molecules have the stoichiometric composition of C2H5N or HCNO: ethylenimine, N-methyl methanimine, ethenamine, and acetaldimine all have the composition of C2H5N, while isocyanic, cyanic, and fulminic acids have the composition of HCNO. The literature data suggest that the loss of water is favored for a 6,4-dimer while the loss of isocyanic acid may be attributed to the dissociation of the cyclic product.9 CID Experiments with Thymine Dideoxynucleotide. A similar set of experiments was performed with aqueous solutions of thymine dideoxynucleotide d(5′-TT-3′) in the negative ion mode. The CID spectrum measured for the deprotonated dideoxynuclotide (see Figure 2) indicates that the monoanion with m/z 545 derived from solution dissociates via the loss of a neutral most likely corresponding to thymine. However, when the ion with m/z of 545 derived from the irradiated solution of the oligonucleotide was subjected to CID, loss of a neutral corresponding to furfuryl alcohol was observed in addition to the loss of thymine. The presence of this new dissociation channel suggests that an intramolecular bonding event was induced in the thymine dideoxynucleotide d(5′-TT-3′) (m/z 545) by the UV irradiation. DMS/MS Experiments with Protonated Dimers of Thymine Nucleobase. Figure 3 (top) shows the presence of possibly three structural isomers in the DMS spectrum in the absence of a modifier, but two of the peaks overlap significantly. The bottom spectrum of Figure 3 demonstrates the marked improvement in separation of three isobaric species that is achieved upon addition of the water modifier. The peaks all shift toward the negative region of CV, which is commonly observed for the samples exposed to a modified transport gas, and they are completely separated at the baseline. The largest shift in the position of the

Figure 3. Effect of water modifier on the separation of three isobaric positive ions with nominal m/z 253 generated from an electrosprayed solution of thymine nucleobase previously exposed to UV radiation. (See text.) The separation voltage was 2400 V p-p, and the transport gas was composed of nitrogen (top pane) and nitrogen with water modifier (bottom pane). The curves labeled “a” correspond to the MRM transition 253/235 (protonated 6,4-adduct), the “b” curves correspond to the transition 253/210 (protonated cyclobutane adduct), and the “c” curves correspond to the transition 253/127 that is characteristic for the proton-bound dimer. The resolution settings were medium resolution (top pane) and high resolution (bottom pane).

peak was observed for the MRM transition 253/210 (the protonated cyclobutane dimer), and the least shift was observed for the protonated 6,4 covalently bound dimer. The separation into three peaks confirms the presence of three isomers and rules out the presence of an isomer that gives rise to two dissociation products. One of the main conclusions that may be drawn on the basis of these results is that the successful separation of the suggested isomers confirms their presence. We had assumed that the extra dissociation channels exhibited by m/z 253 after the exposure to UV light are due to the presence of two isobaric species. However, our initial assumption could have been wrong. If only one covalently bound species was present that was responsible for both extra fragmentation channels, we would observe a complete overlap of the mobility curves characterizing two transitions from the same parent ion. DMS/MS Experiments with Thymine Dideoxynucleotide. The experimental setup for UV-induced reaction of d(5′-TT-3′) was identical to that that was used for the irradiation of aqueous solutions of thymine. In order to verify our hypothesis regarding the presence of two isomeric species in the solution of irradiated oligonucleotide, a deprotonated oligonucleotide and a deprotonated oligonucleotide in which the nucleobases are bound covalently (see Scheme 2), we selected two MRM transitions, 545/ 419 and 545/447, to be measured in the DMS cell. The top spectrum in Figure 4 already demonstrates that these two transitions, 545/419 and 545/447, are characteristic of two different isomers. The addition of isopropanol to the curtain gas resulted in significantly better-resolved peaks. As in the case of thymine dimers, we observe a shift of the peaks toward the negative region Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

6165

Scheme 2. Schematic Representation of the Proposed Bond Transformation in Deprotonated Dideoxynucleotide after UV Irradiationa

a

Also shown are the predominant CID products before and after UV irradiation.

Figure 4. Effect of the modifier on the separation of two isobaric negative ions with nominal m/z 545 generated from an electrosprayed solution of thymine dideoxynucleotide previously exposed to UV radiation. (See text.) The separation voltage was 3900 V p-p, and the transport gas was composed of nitrogen (top pane) and nitrogen with isopropanol (bottom pane). The curves without asterisks correspond to the MRM transition 545/419 that is characteristic for the deprotonated thymine dideoxynucleotide. The curves that are marked with asterisks correspond to the transition 545/447 (cyclic covalentbound photoproduct). The resolution settings were medium resolution (top pane) and low resolution (bottom pane).

of the compensation voltage. Figure 4 demonstrates that the modified, covalently bound thymine dideoxynucleotide shifted much further than the original deprotonated species. Interpretation of the Shifts in the DMS/MS Spectra. The physical nature of the field mobility dependence in DMS is best 6166

Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

described by the cluster formation model (the dynamic clusterdecluster model)14 according to which the selectivity in the DMS spectra increases upon addition of a modifier due to the interactions that take place between the ionic species and the neutral modifier. Essentially, the shift in the position of the mobility peak as a function of the compensation voltage is determined by these unique interactions of the drifting ions with the neutral modifier. The localization, availability, and absolute value of the charge on the ion all play a role in these interactions. For the protonated thymine dimers that were observed in the positive ion mode with H2O as the modifier, the largest shift in the position of the peak was observed for the cyclobutane isomer (the MRM transition 253/210) and the least was observed for the 6,4 covalently bound dimer (the MRM transition 253/235). The proton-bound isomer (the MRM transition 253/127) exhibited an intermediate shift, slightly larger than that observed for the 6,4 covalently bound dimer. One can speculate that the extent of clustering of these species depends on how much the proton is exposed to possible interactions with the neutral polar modifier (H2O in this particular case). We note that the proton may interact intramolecularly with several basic (high proton affinity) sites on the two thymine molecules of the three isomers of the dimer: the carboxyl oxygens and the primary amino groups. We suggest, taking into account the rigidity of the structure introduced by the presence of the ring, that the proton is exposed the most in the cyclic product ion that exhibits the largest shift of the three isomers upon interaction with the water modifier molecules. The 6,4 product ion is shifted the least, and this suggests that its proton is better shielded from the interactions with water molecules. While the one covalent bond in the 6,4photoproduct still introduces some rigidity into the structure of the dimer, it also brings together two primary amino groups that provide attractive sites for interaction with the proton and

so shielding from the interactions with the neutral modifier. The proton in the proton-bound dimer in which it is shared between the two monomers also may be shielded to a significant extent but apparently less so than in the 6,4photoproduct. One can similarly expect that clustering with isopropanol in the deprotonated d(5′-TT-3′) and its covalently bound isomer is charge directed. However, in this case, the negative charge is localized on the phosphate groups of the ions. The charge on the covalently bound dimer can be expected to be more exposed to the interactions with isopropanol than that on the normal d(5′-TT-3′) isomer. The greater exposure of the charge in the cyclic photoproduct is attributed to the strain imposed by the cyclobutane lesion that makes it difficult for the acidic groups of the ion to interact with its phosphate. (See Scheme 2.) CONCLUSIONS The new analytical technique of differential mobility spectrometry/mass spectrometry (DMS/MS) has been applied to demonstrate the formation of covalently bound isomers of dimers of thymines and of a biomolecule containing adjacent thymines.

Addition of the water modifier was shown to greatly improve the separation of the isobaric species that were studied. On the basis of the results reported here, we can expect that a combination of electrospray ionization tandem mass spectrometry with differential mobility spectrometry should provide a fast, efficient, and sensitive approach to the elucidation of isomeric structures of modified oligonucleotides and DNA generally. ACKNOWLEDGMENT Continued financial support in the form of a research partnership grant from the Natural Sciences and Engineering Research Council of Canada and AB SCIEX is greatly appreciated. As holder of a Canada Research Chair in Physical Chemistry, D.K.B. thanks the contributions of the Canada Research Chair Program to this research.

Received for review April 13, 2010. Accepted June 15, 2010. AC100977B

Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

6167