Structural characterization of melphalan modified 2′-oligodeoxynucleotides by miniaturized LC-ES MS/MS

Abstract

In this study a miniaturized LC coupled to electrospray tandem mass spectrometry was used to analyze modifications originating from the interaction between the chemotherapeutic agent melphalan and 2Ј-oligodeoxynucleotides. Low energy CAD product ion spectra gave information about the specificity of melphalan alkylation with regard to certain DNA sequences. These data can be very useful to estimate the risk in the development of secondary leukaemia as a result of a melphalan cure. In the study of the interaction between melphalan and d(GG), differentiation could be made between alkylation on the 5Ј-side and alkylation on the 3Ј-side, because of the presence or absence of the alkylated w 1 fragment in the low energy CAD spectra. In the other di-mers alkylation specificity for the different bases could be observed. Melphalan alkylation occurs in the sequence G Ͼ A Ͼ C Ͼ T. The study of the alkylated d(GGGG) revealed the presence of mainly 5Ј-end alkylation. Furthermore studies were performed which investigated other melphalan treated di-, tetra-, hepta-, and octa-mers. NA is a target for many xenobiotica and endogenous compounds. Electrophiles from either source can interact covalently with DNA with the formation of so called DNA adducts [1]. These adducts are believed to be the cause for an increased risk of cancer. One of these alkylating agents is the nitrogen mustard melphalan currently used as a chemotherapeutic agent in the treatment of malignant melanoma, multiple myeloma, lymphomas and ovarian carcinoma [2]. DNA adduct formation in perfectly healthy cells at the stage of primary cancer treatment might explain the development of a secondary leukaemia afterwards. It is a well known fact throughout literature that melphalan mainly alkylates the N-7 position in a guanine residue [3]. To the best of our knowledge, no information is available on the regioselectivity of melphalan alkylation in 2Ј-oligodeoxynucleotides and/or whole DNA strands. Therefore there were some important issues left as there are the degree of alkylation of G in relation to its position in the sequence, the influence of the neighbors on the degree of alkylation and the fragmentation patterns observed in melphalan modified 2Ј-oligodeoxynucleotides. Furthermore can we detect C, A and/or T adducts and what is the relation with earlier performed experiments on individual 2Ј-deoxynucleotides and -nucleosides [4,5].Electrospray mass spectrometry has become a viable technique for determining both the sequence specificity and the chemical structure of DNA modifications. By using this technology a lot of information was gathered on the adduct formation between potential carcinogens and individual 2Ј-deoxynucleotides (dNMPs) and -nucleosides (dNs). Furthermore modifications in whole DNA strands are studied after enzymatic digestion also resulting in the study of modified dNMPs and dNs [6]. However, this enzymatic digestion eliminates information related to the location of the adduct in the DNA sequence. In this paper we wish to describe a study of th...

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…Apart from modified nucleobases, LC-ESI-MS also allows for the detection of sugar-phosphate adducts and modified ODNs that are not amenable to GC-MS analysis. [89][90][91] Owing to the highly acidic nature of the phosphate backbone, such analyses are commonly performed in the negative-ion mode. Moreover, the ability of ESI in producing multiply charged ions, in combination with improved mass range of modern mass analyzers, has facilitated the characterization of large analytes like ODNs.…”

Investigation of Enzymatic Behavior of Benzonase/Alkaline Phosphatase in the Digestion of Oligonucleotides and DNA by ESI-LC/MS

“…When coupled to separation techniques such as HPLC and CE, the MS technique is particularly advantageous in analyzing oligonucleotides and DNA adducts in complex mixtures. − The procedure often starts with enzymatic digestion (typically DNase I or nuclease P1) to deoxynucleotides or deoxynucleosides prior to further analysis by MS. ,,, However, DNA sequence information is lost as the oligomers are cleaved to individual deoxynucleotides or deoxynucleosides. It has been observed that reaction of carcinogens with DNA often exhibits sequence selectivity, the recognition of which may be important in order to understand their mutagenic activities. − Hence it is advantageous to be able to obtain the sequence information from the analysis. The ability of electrospray-MS to form multiply charged ions, in combination with improved mass range in modern instruments, has made possible the detection and structural elucidation of larger digested fragments.…”

Quantitative Measurement of DNA Adducts Using Neutral Hydrolysis and LC−MS. Validation of Genotoxicity Sensors

“…Niessen, 9 Andrews et al, 8 and Koc and Swenberg 11 have recently reviewed the analysis of DNA adducts by MS. Applications include quantitation of nucleobase adducts with ethylene oxide, 29 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline, 12 acetaldehyde, 13 ene, 14 urethane, 15 tamoxifen, 16 melphalan, 17 and heterocyclic aromatic amines. 7 DNA adducts formed by oxidative stress were identified using MRM.…”