Novel Analytical Methods for Examining Biomolecular Complexes Using Electrospray Ionization Mass Spectrometry
- Author(s): Flick, Tawnya Grace;
- Advisor(s): Williams, Evan R;
- et al.
Several analytical strategies and investigations are presented in this dissertation to improve the quantification, sensitivity, and structural information that can be obtained for gaseous biomolecular ions in electrospray ionization (ESI) mass spectrometry (MS) experiments. Internal or external standards are commonly employed to quantify molecules in complex mixtures because molecular ion abundances cannot be directly related to the concentration of the molecules in solution. A new standard-free quantitation method is used to obtain the relative concentrations of components in a mixture using the abundances of large, nonspecific clusters formed by ESI. Large non-covalent clusters overcome differences in ionization efficiencies between molecules, and are representative of the solution-phase mixture. The sensitivity in MS experiments can be significantly lowered by the presence of high concentrations of salts in the ESI solution because nonspecific ion adduction to biomolecules distributes ion signal into different forms with various numbers of adducts. Studies here demonstrate the extent of both sodium ion and acid molecule adduction to proteins are inversely related, and both depend significantly on the proton affinity of the anion in the ESI solution. Several solution-phase additives that contain anions with low proton affinity values are shown to effectively desalt protein ions generated by ESI, which should result in improved detection limits, more accurate mass measurements, and improved tandem MS sensitivity. Additionally, a solution-phase additive (HClO4) is discovered that can be used to count the number of basic sites accurately in peptides and proteins based on the number of HClO4 adducts to low charge states. High charge states of peptides and proteins can be readily formed by ESI of aqueous solutions that contain trivalent metal ions, and fragmentation of these trivalent metal ion-peptide or protein complexes by electron capture dissociation can be used to increase the structural information obtained from these experiments. Metal ion-biomolecule interactions are ubiquitous in nature where they play a role in many biological processes. Here, nonspecific metal ion adduction to protein cation and anions is shown to result in more compact conformations compared to the bare protein ion, likely a result of salt-bridge interactions between the metal ion and the biomolecule.