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Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry.

ABSTRACT: An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.

SUBMITTER: Mazzitelli CL

PROVIDER: S-EPMC2531255 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry.

Mazzitelli Carolyn L CL Brodbelt Jennifer S JS

Analytical chemistry 20070518 12

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while elimin ...[more]

PMID: 17508717

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Project description:BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.

Dataset Information

Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry.

Publications

Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry.

Similar Datasets

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