Igor A. Kaltashov
Professor of Chemistry, University of Massachusetts
Ph.D.: University of Maryland Baltimore County Postdoctoral Training: Johns Hopkins University School of Medicine
Mass spectrometry has recently evolved to become one of the major analytical tools to study biomolecular structure and function. "Soft" ionization techniques, such as electrospray ionization (ESI), provide a means to desorb intact biopolymers(proteins, oligonucleotides, polysaccharides, etc.) from solution to the gas phase. Under carefully controlled experimental conditions it is possible in many cases to preserve rather weak non-covalent biomolecular complexes and thus obtain information on binding properties in solution (e.g., protein quaternary structure, enzyme-substrate-cofactor complexes, etc.). On the other hand, rapid progress in designing/refining experimental techniques that are used to fragment mass-selected molecules in the gas phase (tandem mass spectrometry) allows direct determination of covalent structure of large biopolymers (e.g., protein primary structure) in a single experiment. We are utilizing these capabilities of mass spectrometry to study biomolecular architecture and function. Our research is focused on: (a) developing mass spectrometry-based strategies to study higher order structure and folding dynamics of proteins, and (b) applying these techniques to study mechanisms of ligand binding/release mechanism of several transport proteins. We are currently using hydrogen/deuterium (H/D) exchange in solution in combination with ESI MS to probe the conformational stability and folding/unfolding dynamics of proteins in vitro under various conditions. Transiently populated intermediate states are detected and characterized by different degrees of backbone amide protection. Because of the high data acquisition rate, ESI MS offers a facile way to resolve these intermediates on a time scale from tens of msec to hours. The global information on the protein conformational stability and folding dynamics is complemented by local (residue-specific) information. For example, collision-activated dissociation (CAD) of protein ions is used to measure the deuterium content locally as a function of exchange time. This allows us to produce conformational stability maps of both apo- and holo-forms of proteins, thus providing valuable information on the shape of protein folding and binding funnels, from which details of protein function can be deduced. We are also interested in exploring the structure of polypeptide and protein ions in the absence of solvent. Understanding the behavior of bioions in the gas phase is very vital for our research. For example, knowledge of mechanistic aspects of activation and dissociation of protein ions may allow us to "direct" fragmentation to specific sites (protein sub-domains, reduction of disulfide bonds, etc.) in a controlled fashion.