Novel Methods to Study Metastable Biomolecular Systems with Native LC/MS

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With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Prof. Igor Kaltashov and his group at the University of Massachusetts at Amherst are developing powerful analytical tools to study processes such as protein/receptor recognition and enzymatic reactions. These new tools will advance understanding of the mechanisms of complex biological processes at the molecular level, ultimately facilitating manipulation of these processes to achieve desired outcomes. Thus, the new tools can have significant impact not only in chemistry, but also in biotechnology and medicine. Elements of the research involve close interaction with industrial collaborators, adding a valuable dimension to the educational experience of the graduate and undergraduate students involved. Dr. Kaltashov and his group are also engaging in outreach activities which seek to expose local high school students to the state-of-the-art analytical instrumentation at UMass-Amherst.Characterization of biopolymer dynamics and architecture (conformations of individual biopolymer chains and assemblies of multi-unit systems) is both challenging and important for improved understanding of biochemical processes. There is particular need for tools capable of probing transient systems, which are encountered in processes such as protein aggregation, assembly and evolution of quaternary structures, interaction with physiological partners, and enzyme catalysis and transport phenomena. Improved understanding of these phenomena requires reliable kinetics data and knowledge of conformational changes that accompany transitions. In order to improve on existing characterization capabilities, the Kaltashov group is developing a new robust, sensitive, and highly selective experimental strategy that uses a combination of liquid chromatography (LC) run under native conditions (e.g., size-exclusion chromatography) with on-line detection by native electrospray ionization mass spectrometry (MS). The new LC/MS platform incorporates ion manipulation in the gas phase to enable studies of highly heterogeneous systems (e.g., protein-polymer conjugates). Incorporation of on-column H/D exchange and chemical reduction reactions enables top-down HDX MS/MS characterization of higher order structure and conformational dynamics of proteins.