Fundamentals and Applications of Charge Transfer Dissociation Mass Spectrometry

Abstract

The recent surge in the analysis of biomolecules has driven the need for robust, versatile, sensitive, and accurate techniques such as tandem mass spectrometry for compound structural elucidation. Many fragmentation techniques have been developed over the years, but each has their own limitations, such as the loss of post-translational modifications (PTMs), inability to fragment singly charged precursor ions or cost prohibitiveness. To help fill this gap, a novel fragmentation technique called charge transfer dissociation (CTD) has been developed to fragment stored precursor ions through radical-driven fragmentation processes using kiloelectronvolt reagent cations. CTD has shown success in the analysis of proteins, peptides, lipids and oligosaccharides but, until now, the underlying principles behind this fragmentation technique have not been thoroughly explored. This dissertation, therefore, focused on the fundamentals of CTD efficiencies and mechanisms and the extent of structural information that can be gained depending on the charge state and charging adducts of different precursor ions like peptides and oligosaccharides. The first set of experiments investigated the identity of the reagent gas used in CTD and the impact on the sequence coverage and fragmentation efficiency for the analysis of a model peptide, bradykinin and a model oligosaccharide, κ-carrageenan with a degree of polymerization of four (dp4). In past work, CTD employed helium as a reagent gas, but due to the increased scarcity and expense of helium as a consumable, this work explored a variety of alternative reagent gases, including Ar, H2, He, N2, O2, and lab air. Initially, CTD was contrasted with low-energy collision-induced dissociation (LE-CID) for both bradykinin and κ-carrageenan dp4. All of the CTD reagents gases generated near-complete sequence coverage of bradykinin and LE-CID only generated ~56% sequence coverage. For analysis of κ-carrageenan dp4, all the CTD reagents gases generated more structural information than CID, and CTD preserved labile sulfate groups while providing cross-ring cleavages. In contrast, LE-CID spectra contained sulfate losses, glycosidic cleavages and neutral losses. All five reagent gases generated consistent sequence coverage and fragmentation efficiencies relative to He-CTD, which suggests that the ionization energy of the reagent gas has minimal impact on the fragmentation of the biological ions. The majority of the activation energy for bradykinin and κ-carrageenan dp4 comes from the electron stopping mechanism, which involves long-range coupling of reagent cations and electrons bound in the highest occupied molecular orbitals (HOMOs) of the biological ions. Based on these results, any of the alternative reagent gases tested can