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

Abstract

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a n ' and w n ') produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w n ' ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an "in situ" derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5'-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3' and 5' fragments (w nϪm , b m , a m , d mϪ1 ) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site. . Thus far, mass spectrometry has played a minor role in locating abasic sites. Two recent reports show that the use of enzymes to release small oligodeoxynucleotides followed by simple mass measurement [2,3] can be an effective approach to locating abasic sites. Others used enzymes to produce abasic sites and made use of the ease of alkaline cleavage at those sites to produce small ODNs, which can be mass-measured by matrix-assisted laser desorption ionization (MALDI) mass spectrometry [4]. Another recent development of a physical method for detecting abasic sites on DNA is atomic force microscopy [5]. Despite these advances, there remains a need to develop alternative methods that make fuller use of the capabilities of mass spectrometry and that can be used for ODNs either taken as models in studies of DNA damage or as fragments released from damaged DNA.One approach would be to implement tandem mass spectrometry, which is now emerging as an effective tool for structural analysis and quantification of modified ODNs [6 -16]. Vouros and co-workers [11], in an early example, showed that the combination of electrospray ionization (ESI) coupled with tandem mass spectrometry (MS/MS) can determine the molecular mass and the sequence of short, modified ODNs. That group later investigated the chemo selectivity of a carcinogenic diol metabolite on reaction in vitro with an ODN dodecamer [12]. We recently demonstrated that ESI MS/MS and MALDI PSD are successful strategies to distinguish both normal and UV photo-damaged ODNs containing 4 -8 nucleotides [13,14]. 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 inform...

Tandem Mass Spectrometry-Based Detection of C4′-Oxidized Abasic Sites at Specific Positions in DNA Fragments

“…more than normal a-B ions. The proposed structure of the a* ion is shown in Figure 2A and the fragmentation at C4-AP is consistent with that seen in AP-containing oligonucleotides (51). Based on the preferential fragmentation at C4-AP sites and the characteristic mass of the a* ion (18 a. m. u. more than the corresponding a-B ion), the position of the C4-AP sites in an oligonucleotide can be easily determined.…”

Structure Determination of an Interstrand-Type Cis-Anti Cyclobutane Thymine Dimer Produced in High Yield by UVB Light in an Oligodeoxynucleotide at Acidic pH

“…This method employs phosphodiesterase type I and II enzymes to degrade ODNs from the 3′- and 5′-directions respectively, and MALDI to assay the fragments formed 23-26. This method can map abasic sites and adjacent photodimers, both of which terminate the progressive enzymatic cleavage 27-29…”

Automated coupling of capillary-HPLC to matrix-assisted laser desorption/ionization mass spectrometry for the analysis of small molecules utilizing a reactive matrix

“…In that study, however, derivatization was performed off-line as a preliminary step before mixing the sample with the matrix and deposition onto MALDI targets. To our knowledge, only one attempt of utilizing the reactivity of the chosen MALDI matrix was previously made by Zhang and Gross [22] where the anthranilic acid matrix formed Schiff bases with abasic sites of DNA strands.…”