| Cellular proteins often function through their direct interactions with ligands. A ligand could be an atom, a small molecule, DNA/RNA, or another protein that interacts with specific sites of a particular protein. In the solution phase, a ligand interacts through hydrophobic and electrostatic interactions, hydrogen bonding, and van der Waal's interactions. The characterization of protein-ligand interactions (binding affinity, stoichiometry, structures, and sites of binding) is important for future drug design studies. Although a number of biophysical methods are available for protein-ligand analysis, electrospray ionization mass spectrometry (ESI-MS) offers strong advantages in speed, specificity, sample size, and sensitivity for studying such noncovalent complexes. ESI-MS has been applied as a drug screening and chemical biology tool in the pharmaceutical industry because of its ability to measure macromolecules without disrupting covalent bonds while maintaining the weak noncovalent interactions. However, how the absence of water may affect binding affinity, binding sites, protein-ligand interactions, and complex structure are unknown. Thus, the relationship between intermolecular interactions present in the solution phase and the gas phase is not well characterized.;My thesis focuses on developing new top-down mass spectrometry methods for the characterization of protein-ligand complexes. We attempt to address some of these questions in our investigation of gas phase electrostatic and hydrophobic interactions in the protein ribonuclease A and S complex with nucleotide ligands. We use tandem mass spectrometry (MS/MS) to characterize protein-nucleic acid complexes and to identify the putative binding sites of nucleotides to kinases. We also show that MS/MS coupled with a newly developed fragmentation method, i.e., electron capture dissociation (ECD), is suitable for detecting and characterizing weakly bound complexes such as alpha-synuclein protein binding to copper, cobalt, and spermine.;My research has highlighted some important differences between the gas phase and the solution phase, such as the weakening or absence of hydrophobic interactions in the gas phase and the potential strengthening of electrostatic interactions upon removal of water. The latter feature can be exploited by mass spectrometry to determine the sites of ligand binding to targeted proteins. |
