Analysis of M1G-dR in DNA by Aldehyde Reactive Probe Labeling

15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this ..... m/z 188 and m/z. 640f m/z 193, were used to monitor ARP-M1G-dR an...
0 downloads 0 Views 247KB Size
Download PDF
Chem. Res. Toxicol. 2005, 18, 51-60

51

Analysis of M1G-dR in DNA by Aldehyde Reactive Probe Labeling and Liquid Chromatography Tandem Mass Spectrometry Yo-Chan Jeong, Ramiah Sangaiah, Jun Nakamura, Brian F. Pachkowski, Asoka Ranasinghe, Avram Gold, Louise M. Ball, and James A. Swenberg* Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, North Carolina 27599-7431 Received May 26, 2004

A novel method for the measurement of pyrimido[1,2-a]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) f [M1G + H]+ (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing (15NH4)2SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 µg of blank DNA, 23 ng of IS DNA (400 fmol of 15N -M G-dR), and AS DNA containing 0-810 fmol of M G-dR. With the use of this novel IS 5 1 1 DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M1G adducts/108 guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.

Introduction The role of endogenous DNA damage in genetic diseases including cancer is an important issue in current research. Reactive oxygen species (ROS)1 include endogenous agents capable of causing multiple types of DNA damage through different pathways. Estimations of such oxidative DNA damage range from 10000 to over 200000 events per cell (1, 2). ROS can directly react with DNA bases or deoxyribose, producing modified bases, abasic sites, and DNA strand breaks (3). While base oxidation and abasic sites are the most frequent insults to DNA, substantial evidence supports that secondary DNA damage produced from byproducts of primary DNA damage or lipid peroxidation may play an important role in * To whom correspondence should be addressed. Tel: 919-966-6139. Fax: 919-966-6123. E-mail: [email protected]. 1 M G, pyrimido[1,2-a]purin-10(3H)one; M G-dR, 3-(2′-deoxy-β1 1 D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)one; ARP, aldehyde reactive probe (N′-aminoxymethylcarbonylhydrazino D-biotin); SPE, solid phase extraction; SRM, selective reaction monitoring; AS, analytical standard; IS, internal standard; CTD, calf thymus DNA; MDA, malondialdehyde; E. coli, Escherichia coli; ROS, reactive oxygen species; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; MSM, minimum salt medium; COSY, correlated spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation; NOESY, nuclear overhauser effect spectroscopy.

mutation (4, 5). Base propenal, a byproduct of deoxyribose oxidation, has been identified as a key intermediate, producing the tricyclic DNA adduct, pyrimido[1,2-a]purin-10(3H)one (M1G) (6). More frequently, ROS can abstract a hydrogen from polyunsaturated fatty acids to produce a carbon-centered fatty acid radical, which can be further oxidized to form a lipid peroxy radical. Lipid peroxy radicals abstract hydrogen from neighboring phospholipids, producing additional lipid peroxy radicals. As a result, there may be up to 2 orders of magnitude amplification in the production of free radicals (7, 8). Lipid hydroperoxides are degraded to multiple reactive byproducts such as malondialdehyde (MDA), 4-hydroxynonenal, crotonaldehyde, and acrolein, which can damage genomic DNA (9). Lipid peroxidation-induced DNA adducts include tricyclic purine adducts such as 1,N6ethenodeoxyadenosine, N2,3-ethenodeoxyguanosine, and 3-(2′-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)one (M1G-dR) (10). MDA, which has been extensively used as a biomarker for lipid peroxidation, is the most mutagenic byproduct. β-Hydroxyacrolein, a tautomer of MDA, is highly reactive toward guanine, producing M1G (11). Because of the continuous production of ROS and MDA during lipid peroxidation chain reactions, a relatively high number of M1G-dR lesions have

10.1021/tx049853l CCC: $30.25 © 2005 American Chemical Society Published on Web 12/09/2004

52

Chem. Res. Toxicol., Vol. 18, No. 1, 2005

been reported in animal and human tissues (12, 13). In human pancreatic tissue, the level of M1G in the DNA exceeded that of 8-oxoguanine, the most commonly measured form of oxidative DNA damage. Therefore, it is imperative to accurately measure M1G to determine the biological significance of M1G in disease (14). Attempts to analyze M1G have been hampered by its multiple chemical forms that change during DNA analysis and hydrolysis. When the adduct is present in doublestranded DNA, base pairing of dC opposite M1G facilitates ring opening to N2-oxopropenyl-dG (15). The aldehydic moiety in N2-oxopropenyl-dG is reactive toward amine compounds (15, 16), which can hinder accurate measurements due to competitive reactions that produce unstable conjugates (17). Furthermore, the ring-opened form is frequently not recognized by the anti-M1G antibody that has been used in immunoaffinity purification or immunoslot-blot measurement (2). In the present study, a stable conjugate of M1G was generated by reaction with aldehyde reactive probe (ARP) (Scheme 1). ARP has been successfully used to produce stable conjugates with ring-opened, aldehydic AP sites for measurement by a slot blot method (18, 19). The strategy of this research for measuring M1G-dR consisted of producing stable ARP-M1G-dR conjugates in DNA prior to enzymatic hydrolysis of the DNA to the nucleoside level and subsequent quantification of ARP-M1G-dR by mass spectrometry. With several major instrumental improvements during the past decade, mass spectrometry has gained more popularity in DNA adduct analysis due to its outstanding specificity and sensitivity (20-23). Application of a stable isotope-labeled DNA adduct as an internal standard (IS) dramatically enhances the accuracy of adduct measurement (24). However, the accuracy and precision of mass spectrometry can be undermined if the use of IS bases or nucleosides cannot account for artifacts that arise during DNA hydrolysis, such as additional oxidation of unmodified nucleotides; incomplete hydrolysis of DNA to nucleosides; or interactions between DNA adducts and other molecules. In addition, M1G is hydrolyzed to a ringopened aldehyde form by base pairing with dC, which does not happen to the free M1G base or its nucleoside under the same conditions. This behavior can limit the use of M1G base or M1G-dR as a standard in analysis. Furthermore, measurement is affected by the competitive reaction of the aldehyde moiety of the ring-opened form with amines as well as differences in hydrolysis efficiency. To overcome these pitfalls, M1G analytical standard (AS) and IS were prepared from high purity genomic DNA and 15N-labeled DNA, respectively. This approach mimics M1G-dR in sample DNA and is expected to produce reliable results that account for artifacts that can arise during sample analysis.

Materials and Methods Materials. HPLC grade solvents were purchased from Fisher Scientific (Raleigh, NC). ARP was purchased from Dojindo Molecular Technologies (Gaithersburg, MD). trans-N-Deoxyribosylase was a generous gift from F. Peter Guengerich (Vanderbilt University, Nashville, TN). 15N-ammonium sulfate was purchased from Spectra Stable Isotopes (Columbia, MD). Unless stated otherwise, all other chemicals and enzymes were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and were used without further purification. Instrumentation. HPLC analysis to characterize the ARPM1G-dR conjugate was performed using a Hewlett-Packard

Jeong et al. 1040A diode array detector combined with an Agilent Technologies (Palo Alto, CA) 1100 Series quaternary pump and degasser. The reaction mixtures were separated on C-18 Platinum EPS column (Alltec, Deerfield, IL, 150 mm × 4.6 mm, 5 µm). Full scan and tandem mass spectra were obtained using a Finnigan (Woburn, MA) LCQDECA ion trap mass spectrometer. Quantification of ARP-M1G-dR and its IS were performed using a Finnigan Quantum triple-quadrupole mass spectrometer connected to a Finnigan Surveyor Micro-LC. NMR data were obtained on a Varian (Palo Alto, CA) INOVA 500 spectrometer at 500 MHz for 1H and 13C NMR at 125 MHz. NMR spectra were recorded in DMSO-d6 at ambient temperature. 1H and 13C chemical shifts and coupling constants were determined by spectral simulation using MestRe-C 3.5.1 software ([email protected]). M1G, M1G-dR, and ARP-M1G-dR Synthesis. M1G and M1G-dR were prepared by the reaction of guanine with tetramethoxypropane according to a published procedure (25) with slight modification. Ten millimoles of tetraethoxypropane was added dropwise to 50 mM guanine in 200 mL of 1 M hydrochloric acid. After 30 min at 70 °C, the reaction mixture was cooled to 0 °C on ice. Excess guanine was precipitated after the addition of K2CO3 to adjust the reaction mixture to neutral pH. After centrifugation at 3000 rpm for 20 min, the supernatant was divided into two parts for purification of M1G or synthesis of M1G-dR. To isolate M1G, the reaction mixture was lyophilized, and then, the remaining solids were extracted twice with methanol followed by concentration of the extract to 1 mL using a vacuum evaporator. M1G was purified from the concentrate by column chromatography on silica gel with methanol:chloroform (12:88) elution. M1G-dR was prepared by incubating the crude M1G fraction with the same volume of aqueous solution containing trans-N-deoxyribosylase (400 µg), 5 mmol of deoxycytidine, and 100 mmol of MES. The enzymatic glycosylation of M1G proceeded for 2 h at 37 °C. The reaction mixture was lyophilized and extracted with methanol for subsequent column chromatography with silica gel. M1G-dR standard was eluted with methanol:dichloromethane (8:92). M1G and M1G-dR standards were characterized by electrospray ionization mass spectrometry (ESI-MS) and 1H NMR (data not shown). To test the reactivity of ARP toward M1G-dR, equimolar (1 mM) M1G-dR and ARP were incubated in 1% acetic acid solution for 30 min at 70 °C. The reaction mixture was separated by HPLC. Three fractions (conjugates 1, 2, and 3; Figure 1) were collected and reanalyzed by HPLC. For the scaled-up synthesis, 9 mg of M1G-dR was dissolved in 6 mL of 0.2% acetic acid solution followed by the addition of 6 mL of 10 mM ARP solution. The reaction mixture was incubated for 2 days at 4 °C. The precipitate was collected by centrifugation and washed twice with 6 mL of cold water. The precipitate was lyophilized to remove any remaining water (recovery, 13.6 mg). 1H NMR (500 MHz, DMSO-d6): 11.16, 11.05 (2 × bs, 1H total, NH9syn/anti), 9.77 (bs, 0.5H, NH15syn/antiARP), 9.74 (bs, 1H total, NH14syn/antiARP), 9.73 (bs, 0.5 H, NH15syn/antiARP), 9.61 (d, 0.5H, NH5syn), 9.49 (d, 0.5H, NH5anti), 8.10, 8.06 (2 × s, 1H, H2syn/anti), 7.98 (d, 0.5 H, J7,8 ) 10.2 Hz, H8anti), 7.60 (dd, 0.5 H, J6,7 ) 14.1, J6,5 ) 11.1 Hz, H6syn), 7.52 (dd, 0.5 Hz, J6,7 ) 13.9, J6,5 ) 10.8 Hz, H6anti), 7.28 (d, 0.5 H, J7,8 ) 9.8 Hz, H8syn), 6.41 (bs, 1H, NH3ARP), 6.35 (bs, 1H, NH1ARP), 6.33 (dd, 0.5 H, J7,6 ) 14.1, J7,8 ) 9.8 Hz, H7syn), 6.24 (dd, 1 H, J1′,2′′ ) 14.1, J1′,2′ ) 7.3, H1′), 5.82 (dd, 0.5 H, J7,6 ) 13.9, J7,8 ) 10.2 Hz, H7anti), 5.31 (bs, 1 H, OH3′), 4.90 (bs, 1 H, OH5′), 4.47, 4.43 (2 × s, 1 H total, CH217syn/antiARP), 4.39 (bs, 1 H, H3′), 4.30 (m, 1 H, H8ARP), 4.11 (m, 1 H, H4ARP), 3.83 (m, 1 H, H4′), 3.56-3.58 (m, 2 H, H5′,H5′′), 3.09 (m, 1 H, H5ARP), 2.82 (dd, 1 H, Jgem ) 12.5, J7cisARP,8ARP ) 2.8 Hz, H7transARP), 2.58 (m, 1 H, H2′′), 2.57 (bd, 1 H, Jgem ) 12.5 Hz, H7cisARP), 2.12 (bt, 2 H, JCH211ARP,CH212ARP ) 6.5 Hz, CH212ARP), 1.62 (m, 1 H, CH29diastereotopicARP), 1.53 (m, 2 H, CH211ARP), 1.47 (m, 1 H, CH29diastereotopicARP), 1.33 (m, 2 H, CH210ARP) ppm. 13C (125 MHz, DMSO-d6): 169.8 (C13ARP), 167.0 (C16ARP), 161.6 (C2ARP), 152.1 (C8anti), 149.0 (C8syn), 148.6 (C3a), 137.3 (C2), 135.9 (C6syn), 134.8 (C6anti), 117.7 (C10a), 102.4 (C7anti), 98.7 (C7syn), 87.9 (C4′), 83.1 (C1′), 71.1 (C3′), 61.1 (C4ARP), 59.2 (C8ARP), 55.5 (C5ARP), 39.9

Analysis of M1G-dR in DNA

Chem. Res. Toxicol., Vol. 18, No. 1, 2005 53

Figure 1. HPLC-UV analysis of the reaction mixture of M1G-dR and ARP. An equal molar ratio of ARP and M1G-dR was incubated in a weak acid solution. The resulting mixture was separated by the HPLC connected to a photodiode array detector. (C7ARP), 39.6 (C2′), 32.9 (C12ARP), 27.1 (C10ARP), 27.0 (C9ARP), 24.3 (C11ARP) ppm. Blank DNA Preparation for M1G-dR Assay. High quality genomic DNA was prepared from snap-frozen calf thymus tissue (26). The frozen tissue sample (2 g) was homogenized in 50 mL of lysis buffer containing 10 mM HEPES (pH 7.4), 1 mM EDTA, 1% SDS, and 2 mM 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Proteinase K was added to the homogenate at a final concentration of 8 units/mL and incubated in a cold room (4 °C) overnight. Protein was precipitated by the addition of onethird volume of protein precipitation solution (Promega, Madison, WI) followed by centrifugation at 4000g for 20 min. The nucleic acid was isolated from the supernatant by the addition of an equal volume of 2-propanol. The precipitated nucleic acid pellet was washed with 70% ethanol and reconstituted in 45 mL of 10 mM HEPES buffer (pH 7.4) with 0.2 mM TEMPO. RNase A (36 KeU) and RNase T1 (12.5 kU) were added to the nucleic acid solution, and the mixture was incubated for an hour at 37 °C. DNA was precipitated by the sequential addition of 2.25 mL of 4 M NaCl and 90 mL of cold ethanol. The DNA was washed once with 90 mL of 70% ethanol solution. Following removal of the ethanol solution by centrifugation, the precipitated DNA was dried on ice for 15 min. The DNA pellet was reconstituted in 30 mL of HPLC grade water. The DNA concentration was determined using UV absorbance at 260 nm in TE buffer (pH 8.0). The DNA yield was 46 mg. To prepare M1G-dR blank DNA, endogenous M1G-dR in calf thymus DNA (CTD) was reduced to 5,6-dihydro-M1G-dR. Briefly, 15 mL of CTD solution (23 mg) was mixed with an equal volume of an aqueous solution containing 10 mM hydroxylamine and 20 mM sodium cyanoborohydride. The reaction mixture was incubated for an hour at 45 °C with mild agitation. After the reaction mixture cooled to room temperature, the DNA was precipitated by adding NaCl and ethanol as described above. The DNA pellet was washed with 70% ethanol and reconstituted in 45 mL of water. To remove any remaining reducing reagent, DNA was precipitated once again using ethanol. Following washing with 70% ethanol to remove NaCl, the DNA pellet was dried briefly on ice and reconstituted in 15 mL of water followed by storage at -80 °C in small aliquots (500 µL) until it was ready to be used. M1G-dR Assay Standard and IS DNA Preparation. Stable isotope-labeled DNA containing 15N5-M1G-dR was prepared from Escherichia coli cultured in minimum salt medium (MSM) containing (15NH4)2SO4 as a single nitrogen source for bacterial growth. 15N-MSM was prepared according to a published procedure (27) with minor modifications [(15NH4)2SO4, 700

mg; KH2PO4, 1.6 g; Na2HPO4, 5.3 g; glycerol, 20 g; MgSO4‚7H2O, 300 mg; Na-citrate‚2H2O, 570 mg; CaCl2, 0.566 mg; Na2EDTA, mg; FeCl3‚6H2O, 25 mg; CuSO4‚5H2O, 0.24 mg; MnSO4‚H2O, 0.127 mg; ZnCl2, 0.0128 mg; and CoCl2‚H2O, 0.494 mg dissolved into a final volume of 1 L]. E. coli (DH5R F′IQ, Invitrogen, Carlsbad, CA) was inoculated by spatula in 1 mL of 15N-MEM followed by culturing for 2 days at 37 °C in an environmental shaker. One milliliter of fresh medium was mixed with 30 µL of bacterial culture for subculturing. After two additional subcultures, 100 µL of the bacterial culture was transferred to 1 L of fresh 15N-MSM. After incubation for 3 days, the bacteria were collected by centrifugation for 20 min at 6000g. Lysis buffer (40 mL) was added followed by homogenization for DNA isolation. The 15N-labeled DNA was isolated from the bacteria by the protocol used for CTD isolation. The recovery was 6 mg based on UV absorbance measurement. To induce a high number of M1G adducts in the DNA, 15N-labeled DNA or CTD was treated with MDA for IS DNA or AS DNA, respectively. MDA was prepared from tetramethoxypropane in 1 M HCl by hydrolysis at 45 °C for 45 min. Three milligrams DNA was incubated for 2 h in a cold room (4 °C) with 6 mL of an aqueous solution containing 50 mM MDA. Unreacted MDA was removed by two precipitations of the DNA with ethanol as described above. Additionally, the DNA was washed with water several times on a Centricon-100 microfilter (Millipore, Billerica, MA) to remove any remaining MDA, salt, and small DNA fragments. To determine the M1G number in AS or IS DNA, 30 µg of DNA was hydrolyzed in 150 µL of 10 mM HCl solution at 70 °C for 30 min. The depurinated DNA bases were separated by HPLC on a Platinum EPS column (5 µm, 4.6 mm × 150 mm) and monitored by UV. M1G was monitored with fluorescence (Ext, 363 nm; Emt, 500 nm) by a LS-40 fluorescence detector (Applied Biosystems, Foster City, CA), and the M1G concentration was calculated based on a standard calibration curve. Total DNA concentration was calculated based on Gua and Ade measured by UV absorbance at 254 nm after separation by HPLC to give the ratio of M1G-dR to nucleotides in the standard DNA. Bleomycin Treatment of DNA. Aqueous solutions containing different amounts of bleomycin with the same molar ratio of ferrous ammonium sulfate were added to a CTD solution (final concentration, 0.3 mg/mL) in 50 mM potassium phosphate buffer (pH 7.4). The mixture was incubated for 2 h at 37 °C with mild agitation. The incubation was terminated by transferring the sample to ice. DNA was precipitated with ethanol and then washed extensively with water on Centricon-30 microfilters following ethanol precipitation.

54

Chem. Res. Toxicol., Vol. 18, No. 1, 2005

Jeong et al.

Analysis of M1G-dR in DNA. Sample or blank DNA (200 µg) with a known amount of M1G-dR was mixed with IS DNA corresponding to 400 fmol of 15N5-M1G-dR. The DNA mix was incubated with 2 mM ARP in 0.1% acetic acid solution for 30 min at 50 °C. Aliquots (10 µL) were subjected to mild acid hydrolysis followed by HPLC-UV measurements of purine base to determine DNA concentration in the sample mix. Unbound ARP in the sample solution was removed by ethanol precipitation of DNA followed by reconstitution in 450 µL of 20 mM HEPES buffer (pH 7.8) containing 1 mM MgCl2. The DNA was hydrolyzed to nucleosides by DNase I treatment at a final concentration of 100 U/mL for 20 min at 37 °C, followed by the addition of a mixture containing alkaline phosphatase, phosphodiesterase I, and phosphodiesterase II to final concentrations of 5, 0.06, and 0.1 U/mL, respectively. The DNA and enzyme mixture was further incubated at 37 °C for 1 h with mild agitation. After the removal of the enzymes by microfiltration, the filtrate was placed on an solid phase extraction (SPE) column (HLB-60, Waters, Milford, MA) that had been preactivated with methanol and equilibrated with water. The SPE column was washed once with 1 mL of water and twice with 3 mL of 20% methanol. ARP-M1G-dR and the IS were eluted with 3 mL of 50% methanol in water. The solvent was removed by vacuum evaporation. The samples were reconstituted in 50 µL of 10% methanol in water and stored at -80 °C until analysis. LC-ESI+-MS/MS Analysis. ARP-M1G-dR and IS were separated on a Platinum EPS C18 column (3 µm, 2 mm × 150 mm) at a flow rate of 0.2 mL/min. After sample injection, the column was washed with 5% acetonitrile in 8.3 mM acetic acid for 2 min. The acetonitrile was gradually increased to 50% over 10 min, kept at 50% for 3 min, followed by reequilibration to initial conditions. The LC eluant was delivered to waste for the initial 7 min, and then, the flow was introduced to the mass spectrometer for 2 min followed by diversion to waste until the end of analysis. The analysis was performed by positive ion electrospray using selective reaction monitoring (SRM). Nitrogen gas was used for the sheath and auxiliary gas. The needle spray voltage was maintained at 4.5 kV. The ion skimmer was placed at a 90° angle relative to the spray needle, which was maintained at a voltage of 70 V. The capillary temperature was 350 °C. The first and third quadrupole mass analyzers were used for unit mass resolution, and the second quadrupole was used for ion fragmentation. Fragmentation was accomplished at a collision energy of 48 V using argon gas at a pressure of 1.5 mTorr. Two ion transitions, m/z 635f m/z 188 and m/z 640f m/z 193, were used to monitor ARP-M1G-dR and ARP-15N5-M1G-dR.

Results and Discussion ARP-M1G-dR Conjugates. Analysis of the reaction between M1G-dR and ARP by HPLC indicated three product peaks in addition to unreacted M1G-dR. UV spectra of the HPLC peaks were qualitatively similar, with λmax in the range of 310-320 nm characteristic of aldoximes (Figure 1). This result is consistent with the report of Schnetz-Boutaud et al. (17) who characterized conjugates of hydroxylamine and methoxyamine with M1G-dR. On the basis of HPLC retention times, the products contained in the three peaks were significantly more hydrophobic than M1G-dR, supporting the formation of conjugates with ARP. By full-scan ESI-MS, peak 1 showed ions at m/z 519 and 541, corresponding in massto-charge ratio to the pseudo molecular ion [M + H]+ of a deglycosylated conjugate ARP-M1G and its sodium adduct, respectively. MS/MS of the pseudo molecular ion is consistent with an aglycone, yielding significant daughter ions at m/z 188 and 332 corresponding to [M1G + H]+ and [ARP + H]+ (data not shown). Peak 1 was also identical in UV and mass spectra to the adduct formed

Figure 2. ESI mass spectrum for ARP-M1G-dR conjugate 2. (A) Full scan ESI mass spectrum for conjugate 2. (B) Tandem mass spectrum for parent molecular ion of ARP-M1G-dR + H+ (m/z ) 635).

from direct reaction of M1G with ARP (data not shown). Peak 1 accounted for