High Sensitivity and Fragmentation Specificity in the Analysis of Drug−DNA Adducts by Electrospray Tandem Mass Spectrometry

Abstract

DNA alkylation is a key cellular event in the mechanism of action of clinical anticancer drugs, chemical mutagens, and carcinogens. 1 The nitrogen and oxygen atoms of purines are preferentially alkylated, with the affinity for a particular base depending on the sequence of neighboring bases. It is most likely that the sequence selectivity of the agent is important in determining the nature of the biological response. 2 Methods based on molecular biology have been used extensively to determine the sequence selectivity of alkylation. 3 Such assays are extremely sensitive and may be quantitative; however, they do not yield direct information concerning the structure of the ligand-DNA adducts. NMR spectroscopy 4 and X-ray crystallography 5 enable detailed structural characterization but are time consuming and require large amounts (micromoles) of material. Mass spectrometry should complement these techniques by providing a rapid and sensitive means of determining the sequence selectivity of alkylation and, at the same time, provide some structural detail about the adducts. 6 Several early studies concerned with the sequencing of ligand-DNA adducts by mass spectrometry have involved tandem mass spectrometry (MS/ MS) following fast atom bombardment (FAB) ionization. 7 This approach is limited, however, by the relatively poor efficiency of FAB ionization for oligonucleotides. 8 In contrast, electrospray ionization (ESI) has allowed mass measurement of relatively large oligonucleotides, including modified oligonucleotides, 9 an intact plasmid DNA, 10 and noncovalent drug-DNA complexes. 11 Tandem mass spectrometry following electrospray ionization (ESI-MS/MS) has been used for the location of modified bases within oligonucleotides and for the characterization of synthetic oligonucleotides which contain "unnatural" bases in their sequence. 12 To date, however, there have been no reports of the use of ESI-MS/MS for the sequencing of covalent, ligand-oligonucleotide adducts. 13 Hedamycin (Figure 1) is a naturally-occurring antitumor antibiotic that binds to double-stranded DNA by both intercalation and alkylation. DNA sequencing shows that alkylation occurs exclusively at guanine bases, but with a distinct preference for guanines located in 5′-CGT-3′ and 5′-CGG-3′ sequences. 14 NMR-derived solution structures of hedamycin covalently bound to double-stranded oligonucleotides show that the anthrapyrantrione chromophore of the ligand threads the double helix, placing the two amino sugars in the minor groove and the alkylating bis-epoxide side chain in the major groove. 15 Our previous studies of hedamycin-DNA binding by ESI-MS have confirmed that hedamycin significantly increases duplex stability and provided strong evidence for the formation of specifically, base-paired duplexes in the gas phase. 16 We demonstrate here that ESI-MS/MS can be used to determine unequivocally the sequence selectivity of base alkylation by a DNA-binding antitumor drug, using hedamycin as a model.ESI-MS/MS spectra of multiply-charged anions of u...

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

“…The product-ion spectrum of the [M Ϫ H] Ϫ from T3X (Table 1, X ϭ abasic site) is considerably simpler than that of the corresponding unmodified ODN, as was noted for abasic sites produced in mass spectrometric fragmentation [15,16]. The precursor containing the abasic site gives only the w 3 ' ion at the abasic site, revealing its location (Figure 1) (we use the prime notation for the fragments as a convenience to distinguish the abasic ODNs).…”

“…Vouros and coworkers [15] also reported a similar method to identify and locate an aflatoxin B 1 modified guanine in three isomeric ODN 9-mers. The latter work of Vouros [15] and that of Sheil's groups [16] demonstrated the simple and informative decomposition reactions of abasic sites produced after loss of a base in a mass spectrometric fragmentation. To our knowledge, no one has systematically studied the fragmentation of ODNs bearing "natural" abasic sites (i.e., sites in which the nucleobase reacts in solution and is replaced with an OH group).…”

Electrospray ionisation mass spectrometry of ruthenium and palladium complexes with oligonucleotides

“…During the last decade the development of electrospray ionisation mass spectrometry (ESI-MS) has allowed accurate mass measurements to be performed on relatively large oligonucleotides including plasmid DNA. [15][16][17][18][19] ESI-MS has also been used for the characterisation of covalent [20][21][22][23][24] and non-covalent [25][26][27][28] drug-DNA complexes. For example, ESI-MS studies on DNA bound to the antibiotic hedamycin revealed that the drug selectively binds to specific DNA sequences and significantly increased the stability of duplex DNA in the gas phase.…”

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures

“…Over the years, mass spectrometry has been employed directly or coupled with high-performance separation techniques to characterize a variety of covalent adducts produced by alkylation of nucleic acids in vivo and in vitro [11][12][13][14][15][16][17][18][19]. The combination of direct infusion electrospray ionization (ESI) [20] and Fourier transform mass spectrometry (FTMS) [21] constitutes an ideal platform for the analysis of this type of samples, which affords the benefits of high mass accuracy and resolving power [22].…”