Implementation of ion/ion reactions and tandem mass spectrometry for biomolecule analysis

Analytical mass spectrometry has been widely applied in the field of molecular biology. It provides unprecedented capability in rapid determination of the molecular mass and the structural information for identification and characterization of biomolecules of interests. Instrumentations equipped with unique dissociation and reaction functionalities have been developed to advance the utilities of mass spectrometry for faster and more accurate biomacromolecule characterization all around the world. In this thesis, the capabilities of a quadrupole/time-of-flight (QqTOF) mass spectrometer coupled with ion/ion reaction functionalities for structural characterization of nucleic acid anions has been demonstrated. In order to use gas-phase dissociation and ion/ion reactions to their full potential, unimolecular dissociation of several model systems, such as non-coding RNAs and chemically-modified oligonucleotide-based drugs, has been studied under various dissociation conditions. Factors, such as the observation time-scale, the ion type and the internal energy distribution of the ion can significantly affect the information contents obtained from gas-phase dissociation of nucleic acid. In addition, the efficacy of proton transfer charge reduction for resolving complicated, highly congested fragment ion spectra from highly-charged intact nucleic acids has also been demonstrated. In contrast to the convention biochemical methods, which are time consuming and ineffective in characterizing chemically-modified oligonucleotides and naturally-occurring RNAs with important post-transcriptional modifications, the quadrupole/time-of-flight (QqTOF) mass spectrometer coupled with ion/ion reaction functionalities has been demonstrated to be a powerful platform for top-down analysis of highly-modified nucleic acids. The second part of this thesis discusses novel methodologies to implement gas-phase bio-ions dissociation via electron transfer reactions for both positively-charged peptide cations and negatively-charged oligonucleotide anions. To circumvent difficulties associated with formation of high mass-to-charge ratio electron transfer dissociation (ETD) reagent anions, a strategy is demonstrated for the formation of reagent radical anions via collision-induced dissociation (CID) of the electrospray ionization (ESI)-generated arene carboxylic acids. The radical anions generated via loss of followed by collision-induced dissociation of CO2 from the deprotonated arene carboxylic acid are effective ETD-reagents. On the other hand, for the implementation of ETD for negatively-charged oligonucleotides, reagent radical cations were generated directly from ESI of polycyclic aromatic hydrocarbon (PAH). The reagent ions were proved to be effective to generate oligonucleotide radical anions for both DNA and RNA oligomers via ion/ion reactions. The subsequent collisional-activation of the resulted DNA and RNA radical anions provides complementary sequence information to CID