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43602-G6
Nucleic Acid Fragmentation from Gas-Phase Ion-Electron Reactions
Our research involves development and application of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and MS/MS (i.e., gas-phase fragmentation of molecular ions) for biomolecular structural characterization. We focus on two techniques based on gas-phase ion-electron reactions: electron capture dissociation (ECD) and electron detachment dissociation (EDD). The former involves irradiation of multiply positively charged ions (produced by electrospray ionization) with low energy electrons and the latter involves irradiation of multiply negatively charged ions with medium (>10 eV) energy electrons. Because these processes trigger radical ion chemistry, complementary fragmentation channels are accessed compared to traditional MS/MS approaches.
In research supported by the Petroleum Research Fund, we explore the radical ion chemistry induced by ECD and EDD for structural determination of nucleic acids. During the past two years, we have performed the first application of these two techniques to the characterization of oligoribonucleotides (RNA) and found both similarities and differences compared to our previous work with oligodeoxynucleotides. These experiments were performed by graduate student Jiong Yang and the resulting data have been published in the Journal of the American Society for Mass Spectrometry. A detailed characterization of ECD and EDD fragmentation pathways is needed before these techniques can be applied to unknowns. Therefore, Jiong also undertook mechanistic studies, including influence of electron energy, number, energy distribution, and ion charge state. These data were included in a manuscript that is currently being revised for the Journal of the American Society for Mass Spectrometry. Jiong also performed double resonance experiments in which ions at a particular ICR frequency are ejected from the ICR cell during the fragmentation event. This approach provides information about reaction intermediates. A manuscript describing these data has been submitted to Journal of Physical Chemistry B. Furthermore, Jiong performed ECD and EDD of chemically modified oligonucleotides used for antisense applications and found that EDD appears more powerful than both ECD and infrared multiphoton dissociation (IRMPD) as it provided full sequence coverage and spectra are straightforward to interpret. A manuscript describing these data is in preparation for Journal of Mass Spectrometry. Jiong successfully defended his Ph.D. thesis, which is largely based on PRF-supported research, on August 29, 2007 and he is now working as FTMS Application Scientist at Bruker Daltonics.
In order to explore the utility of ECD and EDD for characterizing nucleic acid higher order structure, we designed three 15-mer DNAs with different solution-phase structures: two hairpins differing in stability, and one unstructured oligonucleotide. These 15-mers were characterized by EDD, IRMPD, and MS3 involving both those dissociation methods. We found that all three 15mers have higher order structures in the gas phase. Nevertheless, experiments involving EDD yielded different cleavage patterns with less backbone fragmentation for the more stable solution-phase structure as compared to the other two 15mers. By contrast, no major differences were observed in IRMPD. This work was performed by graduate student Jingjie Mo and is published in Analytical and Bioanalytical Chemistry. To further characterize the gas-phase structures of nucleic acids, Jingjie implemented gas-phase hydrogen/deuterium exchange (HDX) experiments in our instrument. From such experiments, she found that the more stable 15-mer consistently exchanges slower than the other two 15-mers, which have very similar exchange rates, correlating with the previous EDD data. Jingjie has extended this work to a range of DNA and RNA hairpins with different loop sizes, stem lengths, and nucleotide composition and found that gas-phase HDX rates correlate with predicted solution-phase stabilities. A manuscript describing these data is in preparation for the Journal of the American Chemical Society. This project also involved first year graduate student Gabrielle Todd who performed a one semester research rotation in my group under Jingjie's supervision. Jingjie passed her data meeting on October 24, 2007 and is planning to defend her Ph.D. thesis in March 2008.
To gain further understanding of the gas-phase HDX process, Jingjie performed a detailed characterization of multiple model oligonucleotides in both positive and negative ion mode. These experiments indicate that oligonucleotide HDX data can be interpreted in light of the previously proposed relay mechanism for mononucleotide exchange. That mechanism involves initial exchange at the phosphate backbone followed by intramolecular exchange, a hypothesis that we tested by investigating the HDX behavior of DNA with a methyl-phosphonate backbone. A manuscript describing the negative ion mode data has been published in Analytical Chemistry and a second manuscript discussing the positive ion mode data is currently in preparation. Finally, second year graduate student, Hangtian Song, has recently begun to explore ECD and EDD of peptide nucleic acids and biologically modified RNA. In summary, the PRF funds have had a strong impact on the PI's career, resulting in three published manuscripts so far, and several others submitted or in preparation. Furthermore, the research supported by PRF has resulted in one Ph.D. thesis and significant progress towards a second one.
