Gas-Phase Nucleic Acid Ion-Electron Reactions: Implementation, Characterization, Fragmentation Pathways, and Mechanistic Aspects.

Abstract

Traditional tandem mass spectrometry, including collision activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD), is valuable for nucleic acid characterization, particularly for chemically modified oligonucleotides, which cannot be effectively characterized by enzymatic techniques. However, one disadvantage is secondary fragmentation, which complicates spectral interpretation and reduces sensitivity. In this dissertation, alternative gas-phase fragmentation strategies involving radical ion chemistry; electron capture dissociation (ECD) and electron detachment dissociation (EDD), are explored for nucleic acid characterization. EDD was implemented for the first time on a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer and shown to yield complementary fragmentation pathways compared to CAD and IRMPD, and higher sensitivity than ECD due to the negative ion mode operation. Optimum fragmentation efficiency was obtained at 16-22 eV. Higher oligodeoxynucleotide charge states provided improved EDD data, presumably due to more extended gas-phase structures. Oligoribonucleotide ECD fragmentation patterns were found to be nucleobase dependent, suggesting that cleavage proceeds following electron capture at the nucleobases. EDD provided complete sequence coverage of hexamer RNAs without nucleobase dependence, suggesting that EDD proceeds following direct electron detachment from the phosphate backbone. FT-IC double resonance (DR) was applied for the first time to characterize IRMPD and EDD fragmentation pathways of oligodeoxynucleotide anions. Results from these experiments suggest that IRMPD proceeds via a similar mechanism as proposed for CAD. Charge reduced radical precursor ions were found to constitute intermediates in EDD of thymidine-containing oligodeoxynucleotides. Furthermore, (a/z minus thymine) ions (a and z type ions correspond to backbone C-O bond cleavage) mainly originate from secondary fragmentation of a/z radical ions for the DNA dT6. ECD and EDD were extended to the characterization of three types of chemically modified oligonucleotides. 2’-methoxy and methylphosphonate modified oligonucleotides underwent limited fragmentation in ECD whereas complete sequence coverage was obtained in most cases from ECD of DNAs containing abasic sites (i. e., a hydrogen has replaced a nucleobase). EDD appeared to be more powerful than both ECD and IRMPD as it provided full sequence coverage and spectra are straightforward to interpret. Overall, EDD shows great promise for analysis of antisense compounds and modified nucleic acids.