Separation and Sequencing of Isomeric Oligonucleotide Adducts

Anal. Chem. 2007, 79, 5312-5321

Separation and Sequencing of Isomeric Oligonucleotide Adducts Using Monolithic Columns by Ion-Pair Reversed-Phase Nano-HPLC Coupled to Ion Trap Mass Spectrometry Wennan Xiong, James Glick, Yiqing Lin, and Paul Vouros*

Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (()-anti-7r,8t-dihydroxy-9t,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene [(()-anti-BPDE] to double-stranded (ds) 5′-PO4--ACCCGCGTCCGCGC-3′/5′GCGCGGGCGCGGGT-3′ oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (()-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards. In recent years, nanospray mass spectrometry (MS) coupled to separation techniques, in particular, nanoflow high-performance liquid chromatography (HPLC)1 or capillary electrophoresis (CE),2,3 has been successfully used to characterize and quantify DNA adducts at the nucleobase, nucleoside, or nucleotide level * Corresponding author. E-mail: [email protected]. (1) Leclercq, L.; Laurent, C.; De Pauw, E. Anal. Chem. 1997, 69, 1952-1955. (2) Gennaro, L. A.; Vadhanam, M.; Gupta, R. C.; Vouros, P. Rapid Commun. Mass Spectrom. 2004, 18, 1541-1547. (3) Ding, J.; Vouros, P. J. Chromatogr., A 2000, 887, 103-113.

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from various sources.4-7 However, such approaches cannot provide any information regarding the site of adduction within the DNA sequence since internucleotide linkages are cleaved when the enzymes digest DNA to individual deoxynucleosides or deoxynucleotides.8 The assignment of the sites of modification by different carcinogens within a target sequence is a very important topic in cancer etiology since isomeric adducts may possess different mutagenic activities due to different biological response to the presence of the adducts. Therefore, the critical part in the analysis of DNA adducts is to develop a method to determine the modification site and frequency on DNA so that one can fully understand the relationship between the chemical behavior of different carcinogens and mutation hot spots. In order to investigate the chemical selectivity in DNA sequences containing multiple target bases competing for adduct formation, positional isomeric oligonucleotide adducts must be separated. Since these compounds differ only in the location of the covalent modification within the given oligonucleotide sequence, their separation poses a significant challenge. Several research groups have reported using HPLC to isolate and purify individual isomeric oligonucleotide adducts first, followed by evaporation of the solvent, removing the salt by solid-phase extraction (SPE) or ion exchange, and subsequent direct infusion by ESI-MS to identify the modification sites.9-11 However, these multiple-step processes are cumbersome, time-consuming, sampleconsuming, and may result in significant sample losses. The analytical method of interfacing LC separations with on-line MS and MS/MS analysis eliminates the need for isolation of individual analytes, decreases the sample requirement, increases the sen(4) Ricicki, E. M.; Soglia, J. R.; Teitel, C.; Kane, R.; Kadlubar, F.; Vouros, P. Chem. Res. Toxicol. 2005, 18, 692-699. (5) Paehler, A.; Richoz, J.; Soglia, J.; Vouros, P.; Turesky, R. J. Chem. Res. Toxicol. 2002, 15, 551-561. (6) Soglia, J. R.; Turesky, R. J.; Paehler, A.; Vouros, P. Anal. Chem. 2001, 73, 2819-2827. (7) Gangl, E. T.; Turesky, R. J.; Vouros, P. Chem. Res. Toxicol. 1999, 12, 10191027. (8) Vanhoutte, K.; Van Dongen, W.; Hoes, I.; Lemiere, F.; Esmans, E. L.; Van Onckelen, H.; Van den Eeckhout, E.; van Soest, R. E.; Hudson, A. J. Anal. Chem. 1997, 69, 3161-3168. (9) Cosman, M.; Ibanez, V.; Geacintov, N. E.; Harvey, R. G. Carcinogenesis 1990, 11, 1667-1672. (10) Ni, J.; Liu, T.; Kolbanovskiy, A.; Krzeminski, J.; Amin, S.; Geacintov, N. E. Anal. Biochem. 1998, 264, 222-229. (11) Marzilli, L. A.; Wang, D.; Kobertz, W. R.; Essigmann, J. M.; Vouros, P. J. Am. Soc. Mass Spectrom. 1998, 9, 676-682. 10.1021/ac0701435 CCC: $37.00

© 2007 American Chemical Society Published on Web 06/19/2007

sitivity, and improves the throughput of the assay. Several groups have coupled conventional HPLC with electrospray tandem mass spectrometry for the analysis of oligonucleotide adducts.12-15 Harsch et al. used LC-ESI-MS/MS to identify the adduct sites as well as the relative binding preferences, following the treatment of a double-stranded (ds) oligonucleotide derived from the hrpt gene sequence, with benzo[c]-phenanthrene diol epoxide.12 Andrews et al. used random endonuclease and benzonase DNA digestion in a nondirectional manner to cleave DNA into oligonucleotides of varying lengths and analyzed the modified oligonucleotides by using a PS-DVB packed column with the ionpairing reagents 1,1,1,3,3,3-hexafluror-2-propanol (HFIP) and triethylamine (TEA).13 This on-line analysis of modified oligonucleotides has been proven to be a better method to elucidate the structure of the formed carcinogen DNA adducts than other off-line methods. However, these methods used 1 mm columns and required a large amount of sample. The major analytical challenge has been to detect levels of DNA adducts at the level of 0.1-1 adducts per 108 unmodified DNA bases with high specificity and accuracy by using only low microgram amounts of DNA in humans exposed to genotoxic carcinogens. The feature of ESI-MS as a concentration-sensitive device makes it compatible with the rapid development of miniaturized HPLC techniques. Moreover, nanospray has emerged as a promising ionization technique in the analysis of biological molecules because of its high ionization efficiency, low flow rates, and high tolerance to salts relative to electrospray ionization. Therefore, coupling nano-LC with nanospray ESI-MS will further increase the mass sensitivity and require even less DNA.8 So far, to the best of our knowledge, there is no report on the use of on-line nano-LC-MS or nano-LC-MS/MS for the analysis of modified isomeric oligonucleotide adducts. In this article, we discuss the performance of a nanoflow system by coupling reversed-phase nano-HPLC using monolithic columns and methanol/water mobile phase containing the ionpairing reagent triethylammonium bicarbonate (TEAB) with nanospray ion trap mass spectrometry in negative ion mode. DNA adducts formed in the reaction of a ds 14-mer oligonucleotide and (()-anti-BPDE in vitro were analyzed. The 14-mer oligonucleotide selected for this study represents the major mutational hotspots codons 157 and 158 in human lung cancers and the benzo[a]pyrene preferential site in the p53 tumor suppressor gene. The goal of this paper was to establish a methodology that had sufficient sensitivity and selectivity to identify not only the location of a modification site in a known gene sequence but also to assess the frequency with which a DNA adduct forms in that sequence using nano-LC-MS/MS. EXPERIMENTAL SECTION Chemicals and Oligodeoxynucleotide Samples Preparation. Caution: (()-anti-BPDE and several of its derivatives are carcinogenic to rodents and should be handled carefully. (12) Harsch, A.; Sayer, J. M.; Jerina, D. M.; Vouros, P. Chem. Res. Toxicol. 2000, 13, 1342-1348. (13) Andrews, C. L.; Yu, C. P.; Yang, E.; Vouros, P. J. Chromatogr., A 2004, 1053, 151-159. (14) Song, R.; Zhang, W.; Chen, H.; Ma, H.; Dong, Y.; Sheng, G.; Zhou, Z.; Fu, J. Rapid Commun. Mass Spectrom. 2005, 19, 1120-1124. (15) Debrauwer, L.; Rathahao, E.; Couve, C.; Poulain, S.; Pouyet, C.; Jouanin, I.; Paris, A. J. Chromatogr., A 2002, 976, 123-134.

Reagents. Acetonitrile, methanol, and water (all HPLC grade) were obtained from Fisher Scientific (Pittsburgh, PA). Triethylammonium bicarbonate (TEAB) and STE buffer were obtained from Fluka (Milwaukee, WI). Divinylbenzene (synthesis grade), styrene (synthesis grade), decanol (synthesis grade), tetrahydrofuran (THF) (analytical reagent grade), azobisisobutyronitrile (synthesis grade), caffeine (analysis grade), and TEA were purchased from Sigma-Aldrich (Milwaukee, WI). Styrene and divinylbenzene were distilled before use. (()-BPDE was obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository (Midwest Research Institute, Kansas City, MO). The synthetic oligodeoxynucleotides were obtained from Integrated DNA Technologies (Coralville, IA). The oligonucleotides were dissolved in water without further purification and were used as stock solutions. The concentration of the stock solution was 0.15 µg/mL. The concentration was calculated from optical density values provided by Integrated DNA Technologies. To obtain ds DNA, equimolar amounts of the two oligonucleotide strands 5′-PO4--ACCCGCGTCCGCGC-3′ (primary strand) and 5′GCGCGGGCGCGGGT-3′ (complementary strand) in STE buffer (10 mM Tris pH 7.8, 50 mM NaCl, 1 mM EDTA) were heated to 10 °C above the melting temperature for 10 min and gradually cooled to room temperature. The completeness of duplex formation was confirmed by HPLC under nondenaturing conditions. (()-anti-BPDE Modified Oligonucleotide Adduct Synthesis. 1 mg of (()-anti-BPDE in 0.5 mL of THF was added gradually over 2 h to a solution of ds 14-mer oligonucleotide in 2 mL of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, pH 7). The reaction mixture was incubated at 37 °C for 48 h. The reaction mixture was extracted three times with 3 mL of chloroform to remove unreacted diol epoxide. The oligonucleotides were precipitated by using a final concentration of sodium acetate of 0.3 M at pH 5.2 in a 70% ethanolic solution. Samples were reconstituted in deionized (DI) water and incubated at 95-100 °C for 10 min, then chilled in a -20 °C freezer immediately to denature. The samples were stored at -20 °C until analysis. High-Performance Liquid Chromatography. An HP1090 series 2 liquid chromatograph was used to generate solvent flow at a rate of 0.2 mL/min (Agilent Technologies, Palo Alto, CA). A microTee (Upchurch Scientific, Oak Harbor, WA) and polyimidecoated fused-silica capillary tubing (2 m length, 360 µm o.d., 50 µm i.d., Polymicro Technologies, Phoenix, AZ) was used to split the flow rate to 200 nL/min, resulting in a split flow ratio of 1000/ 1. The flow from the splitter was then directed to the integrated capillary LC-nano-ESI system. A four-port microbore (0.15 mm) valve (VICI, Valco, Houston, TX) equipped with a 200 nL PEEK internal sample loop was used for manual sample introduction. Monolithic PS-DVB nanocolumns (75 µm i.d. × 360 µm o.d. × 15 cm) were prepared by thermally initiated copolymerization of styrene and divinylbenzene inside a silanized 75 µm i.d. fusedsilica capillary using a mixture of THF and decanol as porogen according to a published protocol16 and optimized as described below to obtain better chromatographic separation. Xterra MS C18 (3.5 µm, Waters, Milford, MA) and PS-DVB particles (3 µm, Hamilton, Reno, NV) were packed into capillary columns in-house (16) Premstaller, A.; Oberacher, H.; Huber, C. G. Anal. Chem. 2000, 72, 43864393. (17) Rozenski, J.; McCloskey, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 200203.

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following a previously published protocol.7 IntegraFrit columns (75 µm i.d. × 360 µm o.d. × 50 cm) (New Objective, Woburn, MA) were packed to a length of 15 cm, cut to a total length of 17 cm (2 cm unfilled), and then conditioned with methanol for 2 h. Each column was used without introducing a fore frit at the head of the packing material or precolumn. The mobile phase was filtered through a 0.22 µm filter before use in the HPLC analysis. A low-pressure in-line filter was placed between the MicroTee and the pump to avoid clogging the spray tip. Separation of (()-BPDE modified oligonucleotides were achieved using a binary mobile phase consisting of 25 mM TEAB, pH 8.3, and methanol. For the first 5 min of each run, the mobile phase was held isocratic at 100% 25 mM TEAB to ensure the sample loop was flushed completely and the analyte was enriched at the head of the capillary column to avoid peak broadening. A gradient was run from 0% to 10% MeOH over 1 min. The methanol was increased linearly from 10% to 50% over 60 min and then stepped to 100% and held isocratic at 100% methanol for 5 min to ensure stable electrospray as the analyte eluted as well as to purge the capillary column of late-eluting species. The capillary column was then allowed to reequilibrate by holding 100% 25 mM TEAB for 45 min prior to subsequent analyses. Electrospray Ionization Mass Spectrometry. ESI-MS was performed on an LCQ Deca quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with a homemade integrated nanospray ion source, which is similar in design to an interface reported previously7 with some modification. The homemade integrated source consisted of a manual VICI valve fitted with a 200 nL internal loop and an XYZ positioner (FP-2 Newport, Irvine, CA). All components were mounted on a rail system fitted to the LCQ. The capillary column was directly connected to a metal-coated fused-silica emitter (distal coated SilicaTips; tubing i.d. 360 µm, o.d. 20 µm, tip i.d. 10 µm, New Objective) by means of a Teflon sleeve (LC Packings, San Francisco, CA) that allowed for a dead-volume-free butt connection. The emitters were cut to short lengths (2 cm) to ensure minimal extra column dispersion. Electrospraying of the nanocolumn effluent was initiated by positioning the tip 3-4 mm from the entrance hole of the heated capillary and applying the voltage directly to the coated tip. A specially designed high-voltage connection adaptor was made inhouse to place the charge directly on the emitter without scratching the metal coating. Both the HPLC and MS instruments were controlled by Xcalibur software version 1.4 (Thermo Finnigan, San Jose, CA). Total ion chromatograms and mass spectra were recorded on a personal computer with the LCQ Xcalibur software. Data from the MS/MS spectra for the adducts was sequenced manually. A syringe pump (Harvard Apparatus, Holliston, MA) equipped with a 250 µL glass syringe (Hamilton, Reno, NV) was used for direct infusion. Mass calibration and coarse tuning were performed in the positive ion mode by direct infusion of a solution of caffeine, methionylarginylphenylalanylalanine, and Ultramark 1621 (all from Finnigan, San Jose, CA). Subsequently, the parameters of the ion optics were tuned at m/z 1394 for maximum ion transmission in the negative ion mode by infusion of 200 nL/min of a 20 pmol/ µL solution of single-strand 14-mer oligonucleotide 5′-ACCCGCGTCCGCGC-3′ in 50:50 (v/v) 25 mM TEAB/methanol. 5314


To analyze the oligonucleotide samples, the mass spectrometer was operated in full scan, negative ion detection mode to assess all masses present. The scan range for these experiments was m/z 150-2000. The samples were analyzed by LC-MS/MS, producing fragmentation data for the modified oligonucleotides. The scan range for the MS/MS spectra was m/z 415-2000. The LC-MS and LC-MS/MS interface conditions were held constant and are as follows: spray voltage, 1.8 kV; capillary voltage, -4 V; capillary temperature, 210 °C; tube lens voltage, -25 V. The LCMS/MS experiments were conducted at a collision width of 3 Da and relative collision energy of 30%. RESULTS AND DISCUSSION Sample Preparation. (()-BPDE modified oligonucleotides were prepared as described above by using the duplex 5′-PO4-ACCCGCGTCCGCGC-3′/5′-GCGCGGGCGCGGGT-3′ (ds sample). Liquid-liquid extractions were performed to remove the unreacted (()-BPDE. The crude reaction mixtures were used directly for analysis without further purification. Mobile Phase Selection. The analysis of nucleic acids by ESIMS suffers from the polyanionic nature of the oligonucleotides and stable adduct ions formed with cations resulting in mass spectra of poor quality. To achieve adequate chromatographic separation of oligonucleotides in reversed-phase HPLC, an ionpairing reagent is often necessary. However, these ion-pairing reagents suppress ionization resulting in deleterious effects on ESI-MS sensitivity, particularly when multiply charged ions are generated. Thus, a compromise had to be found between optimum chromatographic and mass spectrometric conditions. Apffel et al. recommended the use of HFIP adjusted to pH 7.0 with TEA as an ion-pairing reagent and methanol as organic mobile phase, which resulted in efficient HPLC separation of up to 75 bases and high sensitivity.18 However, when we used the HFIP solvent system, although the sensitivity for negative mode detection increased during the analysis, the overall instrument sensitivity dropped after several sample injections due to clogging of the heated capillary by the HFIP. Because of the problems with the HFIP reagent, alternative solvent systems were evaluated. Our group has been using a methanol/10 mM ammonium acetate (pH 8.4) mobile phase system to separate oligonucleotide adducts.19 Initial experiments were conducted using this mobile phase composition; unfortunately this solvent system provided poor chromatographic separation. Solvent systems consisting of and butyldimethylammonium bicarbonate with acetonitrile gradient21 for the HPLC-ESI/MS analysis of oligonucleotides have been reported and were also evaluated. One observation of particular interest was the influence of the composition of the mobile phase on the charge-state distribution in the mass spectrum of the oligonucleotide. The spectrum of the 14-mer oligonucleotide had two dominant charge states, -4 and -3, in the mass range from m/z 100-2000. Ion-pairing reagents combined with acetonitrile are a commonly used buffer system for reversed-phase ion-pair separation of oligonucleotides. However, (18) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325. (19) Harsch, A.; Marzilli, L. A.; Bunt, R. C.; Stubbe, J.; Vouros, P. Nucleic Acids Res. 2000, 28, 1978-1985. (20) Huber, C. G.; Krajete, A. Anal. Chem. 1999, 71, 3730-3739. (21) Mayr, B.; Holzl, G.; Eder, K.; Buchmeiser, C. G. Anal. Chem. 2002, 74, 6080-6087.

when we used acetonitrile as effluent with either ammonium acetate or TEAB, two charge states of -4 and -3 were always detected with the higher charge state predominating in a relative ratio of 2:1. When methanol was used with ammonium acetate, a similar charge state distribution and ratio were observed. On the other hand, when methanol was combined with TEAB, a dramatic shift to the lower charge state (-3) from the higher charge state (-4) was observed with virtually no (-4) charge state present. In addition, the total ion current for the methanol/TEAB solvent system increased nearly 20-fold versus the acetonitrile/TEAB solvent system (data not shown). Therefore, methanol with the ion-pairing reagent TEAB as solvent system was used for all subsequent experiments. Our experience with the HFIP reagent at low concentrations also suggested that the selection of an appropriate ion-pairing reagent concentration was critical. In addition to consideration of the appropriate mobile phase component, the ion-pairing reagent concentration was also evaluated. At high concentrations (50100 mM) ion-pairing reagents have been shown to cause ion suppression due to their low volatility.18 The use of ion-pairing reagent concentrations as low as 10 mM mitigated the ion suppression problem but resulted in unsatisfactory chromatographic separations. However, 25 mM TEAB seemed to be the best ion-pairing reagent concentration to increase the chromatographic separation and MS performance (data not shown). Therefore, 25 mM TEAB was used for all subsequent LC analyses. Emitter Selection. The spray stability in nanospray is controlled by several factors such as the potential difference between the emitter and counter electrode, the emitter tip diameter, and the material comprising the emitter tip. The influence of different nanospray emitters was important for this investigation. Some groups have used a wire inside a capillary for negative ion nanospray which cannot be used for packed columns.22 Other laboratories used the conventional metal or polyaniline-coated nanospray emitters where the metal or polyaniline is coated on the tip. However, due to the increased probability of voltage arcing in negative ion mode, those emitters experienced increased electrical discharge which resulted in the coating being stripped off the silica or glass substrates and tip failure after a short time.23-25 Because nanospray uses very low flow rates, it requires stable electrospray acquisition over an extended period of time. An alternative would be to use an uncoated fused-silica tip connected by a metal liquid junction union employing an upstream electrical contact, which is a method frequently used in nanospray LC-MS. In order to achieve stable and durable nanospray for negative ion mode, three different emitters from New Objective were evaluated: (i) uncoated SilicaTips (tubing i.d. 360 µm, o.d. 20 µm, tip i.d. 10 µm) where the electrical contact was made to the solution through a metal zero-dead-volume liquid junction (Valco, Houston, TX); (ii) metal TaperTips (tubing i.d. 320 µm, o.d. 50 (22) Lim, A.; Prokaeva, T.; McComb, M. E.; O’Connor, P. B.; Theberge, R.; Connors, L. H.; Skinner, M.; Costello, C. E. Anal. Chem. 2002, 74, 741751. (23) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802-3805. (24) Maziarz, E. P., III; Lorenz, S. A.; White, T. P.; Wood, T. D. J. Am. Soc. Mass Spectrom. 2000, 11, 659-663. (25) Bigwarfe, P. M., Jr.; White, T. P.; Wood, T. D. Rapid Commun. Mass Spectrom. 2002, 16, 2266-2272.

µm, tip i.d. 10 µm) where the electrical contact was added directly to the stainless steel tip, and (iii) distal metal coated fused-silica tips (tubing i.d. 360 µm, o.d. 20 µm, tip i.d. 10 µm) which were layered with a conductive coating to provide voltage contact at the end of the tip. The performance of the three different emitters was evaluated by directly infusing the same concentration solution using a Harvard Apparatus syringe pump (Holliston, MA). Figure 1 illustrates the full scan mass spectra for each of the different emitters using a 0.15 µg/mL solution of 5′-ACCCGCGTCCGCGC3′ oligonucleotide with an m/z 1394 [M - 3H]3- in 50/50(v/v) methanol/25 mM TEAB water solution. Parts A, B, and C of Figure 1 represent the uncoated fused-silica, metal TaperTip, and distal metal coated fused-silica emitter tips, respectively. The flow rate during the infusion experiment was 300 nL/min, and 10 spectra were averaged. There was an apparent difference in the spectral quality and the intensity of the ions detected. As shown in Figure 1, parts A-1-C-1, the nanospray signal intensity for a distal coated emitter is approximately 5 times greater than that with metal emitter and 10 times greater than that with the uncoated emitter. In Figure 1, parts A-1-C-1, the y-axis is normalized to the signal in C-1. In Figure 1, parts A-2-C-2, the y-axis is set to 100% relative abundance for the major ion in the spectrum. As can be seen in the figure, when the voltage was applied via a stainless steel liquid junction or directly to the stainless steel metal tips, cation adducts were formed which lowered the sensitivity of the analyte by dispersing the ion abundance among multiple adduct ions. The best sensitivity was obtained by using distal coated emitters due to lower cation adduct formation. Different tip diameters were also evaluated. It was observed that by using smaller i.d. emitters, smaller droplets were generated which yielded more stable nanospray for negative mode with improved desolvation and ionization efficiency (5 µm > 10 µm > 15 µm > 20 µm). However, the 5 µm emitters were easily clogged even when the mobile phase was filtered and with the presence of the in-line filter. The 10 µm emitter was the best choice for our flow rate in order to obtain constant and stable spray. Therefore, 10 µm i.d. metal-coated tips were used for all the subsequent experiments. Once the mobile phase and emitter tip selections had been optimized, the limit of detection (LOD) was evaluated with this optimized system. The LODs for the single-strand 14-mer oligonucleotide were determined using the optimized nano-ESI components and solvents as mentioned above. A 250 nL solution of the sample containing 1.5 ng/mL (3.6 nM) oligonucleotide was injected, which is equivalent to 0.89 fmol loaded on the column, to yield a signal-to-noise (S/N) ratio of 3 under full scan conditions. The limits of detection (LODs) of 14-mer oligonucleotide were 3.6 × 10 -10 M, with an S/N ratio of 3 (equivalent to 89.6 amol loaded on the column) under SIM conditions. Column Selection. The appropriate choice of a stationary phase that provided favorable mass-transfer properties was particularly important for the rapid and highly efficient separation of large molecules with low diffusivities. Conventional microparticulate stationary phases have been applied for the separation of DNA adducts for many years. Dai et al. used a microbore Xterra C18 reversed-phase column with a mobile phase consisting of HFIP and TEA in methanol at 50 °C to separate oligonucleAnalytical Chemistry, Vol. 79, No. 14, July 15, 2007

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Figure 1. Comparison of ion intensities for 5′-ACCCGCGTCCGCGC-3′ oligonucleotide with different emitter tips: (A) uncoated SilicaTips, tip i.d. 10 µm, (B) metal TaperTips, tip i.d. 50 µm, (C) distal coated SilicaTips, tip i.d. 10 µm; (A-1-C-1) normalized y-axis based on the signal for C1; (A-2-C-2) scale relative for each spectrum with the y-axis set to 100% for the most abundant ion.

otides.26,27 Since negative ion mode electrospray requires a basic mobile phase, the Xterra column is well suited to tolerate highpH mobile phase without reducing the column lifetime. Figure 2A-C compares the chromatographic performance obtained with a conventional nanocolumn packed with micropellicular 3.5 µm Xterra MS C18 or 3 µm PS-DVB particles and monolithic PS-DVB column with the same dimension (75 µm i.d. × 360 µm o.d. × 150 mm). Initially, we used a self-packed Xterra MS C18 nanocolumn combined with 25 mM TEAB and methanol solvent system to separate the reaction mixture of 14-mer oligonucleotide-(()BPDE adducts. The selected mass data trace for the modified oligonucleotide is shown in Figure 2A. Poor chromatographic performance was observed presumably due to the interactions between the oligonucleotide and free silanol groups. As a result, this system was not investigated any further. We have previously reported on the use of PS-DVB packed reversed-phase columns to separate oligonucleotide adducts.12 The peaks of the oligonucleotide adducts were partially resolved at (26) Dai, G.; Wei, X.; Liu, Z.; Liu, S.; Marcucci, G.; Chan, K. K. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 825, 201-213. (27) Fountain, K. J.; Gilar, M.; Budman, Y.; Gebler, J. C. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 783, 61-72.


elevated temperature (55 °C) or room temperature. An in-house packed PS-DVB column was used with 25 mM TEAB solution and methanol mobile phase. The result of LC-MS analysis of the reaction mixture of (()-BPDE with ds oligonucleotide is shown in Figure 2B. As observed in Figure 2B, four major peaks eluting between 41.37 and 47.84 min were consistent with the mass of the adducted target sequence ACCCGCGTCCGCGC containing one covalent (()-BPDE modification with a m/z 1521 [M - 3H]3-. Hence, adduct formation must have occurred on at least one of four different sites in the primary strand of the duplex oligonucleotide. The π-π stacking interactions between the aromatic rings of the PS-DVB support material and aromatic ring of the adducted base may partially account for the improved retention and separation of the isomeric polycyclic aromatic hydrocarbon (()BPDE modified oligonucleotides when compared to the Xterra stationary phase. Despite many advantages, the relatively large void volume between the microparticles and the slow diffusional mass transfer for biological macromolecules into and out of the stagnant mobile phase present in the pores of porous stationary phase introduce significant limitations in the separation efficiency of conventional microparticles. One way to overcome these

Figure 2. Capillary IP-RP-HPLC separation of ds (()-BPDE modified oligonucleotide ACCCGCG*TCCGCGC adduct isomers in capillary columns packed with (A) Xterra MS C18, 3.5 µm, 125 Å, 150 mm × 0.075 mm i.d., (B) PS-DVB particles, 3 µm, 100 Å, 150 mm × 0.075 mm i.d., (C) monolithic PS-DVB, 150 mm × 0.075 mm i.d. Mobile phase: A, 25 mM TEAB, pH 8.6; B, MeOH; gradient from 0% to 40% MeOH in 55 min.

limitations is to use monolithic bed, where the separation media consists of a single piece of a rigid macroporous polymer that can easily be prepared and has high chemical stability over a wide pH range. The lack of intraparticular void volume improves mass transfer and separation efficiency. Premstaller et al. have employed IP-RP-HPLC using monolithic PS-DVB capillary columns for the purification of oligonucleotides and ds DNA prior to analysis by ESI-MS.16,28 Both single- and double-stranded nucleic acids ranging in size from 3 nucleotides up to 600 base pairs have been separated and identified. The chromatographic performance of a PS-DVB monolithic column was evaluated by gradient separation of the mixture of 14-mer oligonucleotide BPDE adducts with a gradient of 10-40% methanol and 25 mM TEAB in 55 min. As observed in Figure 2C, the resolution of oligonucleotide-BPDE adducts obtained with the monolithic column was significantly better than that of the nanocolumns packed with either PS-DVB or C18 beads. In order to obtain better column efficiency, the preparation of monolithic columns was modified by adjusting the polarity of the porogen. Monolithic column synthesis requires a pore-forming solvent or porogen which is usually a mixture of two solvents, (28) Oefner, P. J.; Huber, C. G. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2002, 782, 27-55.

macroporogen (more polar) and microporogen (less polar), the ratio of which is modified to obtain optimal pore size. In general, an increase in the polarity of the mixture results in an increase in the average pore size. Here, decanol and THF were used as macroporogen and microporogen, respectively. The polarity of the mixture was modified by changing the ratio between both solvents. The columns used for the separation of the following oligonucleotide adducts were prepared by using a mixture of divinylbenzene, styrene, decanol, and THF with a volume ratio of 5:5:14:1. The chromatogram of the (()-anti-BPDE-modified 14-mer oligonucleotide using the optimized monolithic nanocolumn is shown in Figure 3 and discussed in detail in the next section. Mass Spectrometry. Under the optimized chromatographic, emitter, and column conditions detailed above, LC-MS was employed for on-line separation of in vitro generated isomeric (()anti-BPDE modified ds oligonucleotides. The composite extracted ion chromatogram for the primary strand oligonucleotide from the crude reaction mixture is shown in Figure 3. The unmodified primary oligonucleotide eluted at 13.33 min and was identified by its [M - 3H]3- ion at m/z 1420. The peak eluting later at 17.40 min with m/z 1394 [M - 3H]3- corresponded to the loss of a phosphate group from the unmodified primary oligonucleotide that was lost due to hydrolysis during the BPDE reaction. Nine major Analytical Chemistry, Vol. 79, No. 14, July 15, 2007

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Figure 3. LC-MS composite extracted ion chromatogram for the primary oligonucleotide strand obtained from the injection of a crude reaction mixture of (()-BPDE with ds oligonucleotide on an optimized monolithic PS-DVB nanocolumn: (I) the unmodified 5′-phosphorylated oligonucleotide (m/z 1420), (II) the unmodified 5′-dephosphorylated oligonucleotide (m/z 1394), and (III) the modified 5′-phosphorylated oligonucleotides (m/z 1521).

peaks corresponding to singly modified (()-BPDE oligonucleotides with a m/z 1521 [M - 3H]3- were eluted between 25.16 and 45.82 min. Oligonucleotides containing multiple adducts, which would be expected to elute at longer retention times, were not observed. Subsequent LC-MS/MS analysis was carried out for the ds oligonucleotide adducts. MS/MS fragmentation patterns were obtained for all chromatographic peaks. The online MS/MS spectra for all nine peaks from the primary strand oligonucleotides in the crude reaction mixture are shown in Figure 4. MS/MS spectra were recorded above the half-width of any peak for data interpretation to minimize interference from flanking peaks. A series of fragment ions a - B (derived from the 5′ end of the oligonucleotides) and w (derived from the 3′ end) were used to identify the sites of adduct formation. These fragment ions arose from characteristic decompositions occurring on modified oligonucleotides as described by McLuckey and coworkers.17,29,30 The different fragment ions observed from this MS/ MS analysis are summarized in Table 1, in which the modified oligonucleotides are indicated with an asterisk on the bases bearing the modification. In the MS/MS spectrum of peak III-1 (29) Mcluckey, S. A.; Vanberkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70. (30) Mcluckey, S. A.; Habibigoudarzi, S. J. Am. Soc. Mass Spectrom. 1994, 5, 740-747.


(ACCCGCG*TCCGCGC), for example, the presence of the unmodified fragment series [w2]- to [w7]2- and [a3 - B3]- to [a6 - B6]- as well as observation of the modified fragment ions [w8*]2-, [w10*]2-, [w13*]3-, and [a10* - B10]2-, [a11* - B11]2-, and [a12* - B12]2- indicated that the w-ion arising from the cleavage of the 3′ C-O bond at the seventh base from the 5′ end did not contain the BPDE moiety, whereas w8 contained the BPDE moiety. Therefore the (()-BPDE covalent modification site was located at G7. For each of the remaining peaks, an asterisk marks the position of adduction in the oligonucleotide sequence: peak III-2 (ACCCGCG*TCCGCGC); in the spectrum of peak III-3 (ACCCGCGTCCG*CGC), diagnostic pairs of fragment ions [w3]and [a11 - B11*]2- as well as [w4*]- and [a12* - B12]2- indicated the site of adduction as G11; in the spectrum of peak III-4 (ACCCG*CGTCCGCGC), diagnostic pairs of fragment ions [w9]2and [a5 - B5*]- as well as [w10*]2- and [a6* - B6]- were obtained. Thus, one component of the possible mixture must have had an adduct on G5, which was retained in [w10*]2- and was lost along with G5 upon formation of the [a5 - B5]- fragment. The absence of unmodified [w9]2- and [a5 - B5]- indicated there was no component with an adduct on the G5; in the spectrum of peak III-5 (A*CCCGCGTCCGCGC) fragment ions [w2]- to [w5]- and [w7]2- to [w11]2- were observed indicating BPDE modification at A1, while [a3* - B3]- to [a7* - B7]2-, [a10* - B10]2- to [a13* -

Figure 4. Fragment ion spectra for the modified primary strand oligonucleotides with m/z 1521 [M - 3H]3- ion obtained from online LCMS/MS by monolithic PS-DVB column.

B13]3- were also observed to confirm the A1 modification position; peakIII-6(ACCCG*CGTCCGCGC);peakIII-7(ACCCG*CGTCCGCGC); peakIII-8(A*CCCGCGTCCGCGC);peakIII-9(A*CCCGCGTCCGCGC). There are also some specific fragmentation patterns observed in Figure 4 above which were also reported for modified oligonucleotides29,30 such as m/z 1420 [M* - BPDE]3- corresponding to the loss of the BPDE moiety from an adducted oligonucleotide, m/z 1484.3 [M* - C]3- corresponding to the loss a neutral cytidine base, m/z 1370 [M* - G*]3- representing the loss of the modified G* base for an adducted oligonucleotide, and m/z 1471 [M* - G]3- corresponding to the loss a neutral guanine base. These diagnostic fragments constitute additional data supporting the modification of the G base for peaks III-1 through III-4, III-6, and III-7. Other important diagnostic fragmentation patterns can also be observed for the adenine in the oligonucleotide sequence. For example, m/z 1375 [M* - A*]3- would

correspond to the loss of the modified adenine and m/z 1476 [M* - A]3- would correspond to the loss of an unmodified adenine. Since there is only one adenine in the sequence, the absence of an ion at m/z 1476 [M* - A]3- in the MS/MS spectrum provided additional evidence for the modification of the A base for peaks III-5, III-8, and III-9. Previous work has shown that the exocyclic amino groups of deoxyguanosine and deoxyadenosine nucleophilically attack the C-10 position of (()-anti-BPDE to form four stable covalent diastereomeric adducts each, e.g., (()-trans-N2-BPDE-dG, (()cis-N2-BPDE-dG, (()-trans-N2-BPDE-dA, (()-cis-N2-BPDE-dA (Figure 5).31-34 (31) Jeffrey, A. M.; Jennette, K. W.; Blobstein, S. H.; Weinstein, I. B.; Beland, F. A.; Harvey, R. G.; Kasal, H.; Miura, I.; Nakanishi, K. J. Am. Chem. Soc. 1976, 98, 5714-5715. (32) Meehan, T.; Straub, K. Nature 1979, 277, 410-412.


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Table 1. Assignment of Fragment Ions from MS/MS Collision-Induced Dissociation of m/z 1521 from the On-Line LC-MS Using a Monolithic PS-DVB Columna ion assignment

III-1 m/z

III-2 m/z

III-3 m/z

III-4 m/z

III-5 m/z

III-6 m/z

III-7 m/z

III-8 m/z

III-9 m/z

w2w3w4w5w6w72w82w92w102w112w122w133(a13 - B13)3(a12 - B12)2(a11 - B11)2(a10 - B10)2(a9 - B9)2(a8 - B8)2(a7 - B7)(a6 - B6)(a5 - B5)(a4 - B4)(a3 - B3)(a2 - B2)-

635.1 924.1 1253.2 1542.2 nd 1067.7 1383.2 nd 1692.3 1836.8 1981.3 1417.2 nd 1908.3 1744.3 1599.7 1454.7 nd nd nd 1357.2 1068.2 779.1 nd

635.1 924.1 1253.2 1542.2 nd 1067.7 1383.2 nd 1692.3 1836.8 1981.3 1417.2 nd 1908.3 1744.3 1599.7 nd nd 1975.3 1686.3 1357.2 1068.2 779.1 nd

635.1 924.1 1555.3 1844.3 nd 1218.7 1383.2 nd 1691.8 nd 1980.8 1416.6 nd 1908.3 1592.7 1448.2 1303.7 nd 1975.3 1686.3 1357.2 1068.2 779.1 nd

635.1 924.1 1253.2 1542.2 nd 1067.7 1231.7 1376.2 nd nd nd 1416.6 1368.9 1908.3 1744.3 1599.3 nd nd 1138.7 1988.4 1357.2 1068.1 nd nd

635.1 924.1 1253.2 1542.2 nd 1067.7 1231.7 1376.7 1540.7 1685.2 nd nd 1368.9 1908.3 1744.3 1599.7 1455.7 nd nd 1988.4 1659.3 1370.3 1081.2 792.2

635.1 924.1 1253.2 1542.2 nd 1067.7 1231.7 1376.2 1692.3 1836.8 1981.3 1417.2 nd 1908.3 1744.3 1599.7 1455.7 nd nd nd 1357.2 1068.2 779.1 nd

635.1 924.1 1253.2 1542.2 nd 1067.7 1231.7 1376.2 1692.3 nd 1981.3 1417.2 nd 1908.3 1744.3 1599.7 1455.7 nd nd 1988.4 1357.2 1068.2 779.1 nd

635.1 924.1 1253.2 1542.2 nd 1067.7 1232.2 1376.7 nd 1685.8 nd nd nd 1908.3 1744.3 1599.7 1454.7 nd nd 1988.4 1659.3 1370.3 1081.2 792.2

635.1 924.1 1253.2 1542.2 nd 1067.7 1232.2 1376.7 nd 1685.8 nd nd nd 1908.8 1744.3 1599.7 1454.7 nd 1138.7 1988.4 1659.3 1370.3 1081.2 nd

a

Italics ) ion carrying the covalent modification; bold ) diagnostic ion pairs.

Figure 5. Structures of (()-BPDE and their DNA adducts formed by epoxide ring-opening reaction.

Thus, there are only five residues A1, G5, G7, G11, and G13 within the oligonucleotide sequence under investigation that may provide potential modification sites for which there would be two corresponding peaks for the cis/trans isomers. In addition, there would be two corresponding diastereomer peaks for each cis or trans isomer yielding an expected total of four peaks per target base. (33) Cheng, S. C.; Hilton, B. D.; Roman, J. M.; Dipple, A. Chem. Res. Toxicol. 1989, 2, 334-340. (34) Geacintov, N. E.; Cosman, M.; Hingerty, B. E.; Amin, S.; Broyde, S.; Patel, D. J. Chem. Res. Toxicol. 1997, 10, 111-146.

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Taking this consideration into account, one could deduce based on the MS/MS fragmentation pattern that peaks III-1 and III-2 represent diastereomeric adducts in which (()-BPDE is attached to the same guanine base (G7) of the oligonucleotide. Peak III-3 represents the case in which (()-BPDE adduct is attached to the guanine G11 of the oligonucleotides. Peaks III-4, III-6, and III-7 represent diastereomeric adducts in which BPDE is attached to the same guanine G5 of the oligonucleotide. Peaks III-5, III-8, and III-9 represent diastereomeric adducts in which BPDE is attached

to the same adenine A1 of the oligonucleotide. Surprisingly, no adduct was found in which (()-BPDE is attached to the guanine G13 of the oligonucleotides. The chromatogram depicted in Figure 3 reveals the partial separation of at least nine peaks, with shoulders on some of the peaks indicating the presence of even more compounds. However, no further information could be obtained on the nature of the stereoselectivity of the adducts studied in this work. All data previously obtained on adducts generated from individual nucleosides were obtained using positive ionization mode. Therefore, they could not be used for the interpretation of the fragmentation processes in the present work carried out on oligonucleotides using negative ionization mode since the behavior of the positively protonated species and negative ion toward collisional activation can be totally different. CONCLUSION It is very important to study directly the interaction of activated forms of carcinogen with the specific DNA sequences and identify nucleotide residues which are selectively targeted by these carcinogens. The prime prerequisites that have to be met are high desalting efficiency, low sample requirement, and high analyte recovery. In this regard, an ion-pair reversed-phase nano-LC method using a monolithic nanocolumn coupled to an ion trap mass spectrometer has been developed for the separation and elucidation of diastereomeric oligonucleotide adducts from the reaction mixture of (()-anti-BPDE with selected 14-mer oligo-

nucleotides. By coupling a 75 µm nano-LC column to nano-ESI/ MS, mass sensitivity was improved with very small amounts of biological samples now amenable to analytical characterization at the femtomole level. By using this powerful method, nine isomeric covalent BPDE oligonucleotide adducts were successfully separated and characterized. Although the precise structure of the diastereomeric adducts could not be determined, isomeric differentiation could be achieved using MS/MS experiments on each isomeric adducts. ACKNOWLEDGMENT This work was supported by a Grant (1RO1 CA69390) from the National Institutes of Health. This project was performed, in part, using compound(s) provided by the National Cancer Institute’s Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute, No. N02CB-07008. The authors are also deeply indebted to Dr. Barry L. Karger and his group for providing access to the LCQ Deca instrument and advice on the preparation of monolithic columns. This is Contribution No. 898 from the Barnett Institute.

Received for review January 24, 2007. Accepted May 10, 2007. AC0701435


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Separation and Sequencing of Isomeric Oligonucleotide Adducts

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