Research in the Webb Group

Research in the Webb Group focuses on advancing tools for structural biology and biophysical measurements. Many kinds of proteins and protein complexes have dynamic or disordered regions (or are intrinsically disordered in their entirety) which makes them difficult to study by gold-standard biophysical techniques. We are developing mass spectrometric structural characterization methods that are rapid, accurate, and precise to study these important systems, using the “three S’s” of mass spectrometry: sensitivity, speed, and selectivity. We conduct footprinting, crosslinking, and other bioconjugation experiments coupled with MS detection.

Ongoing Research Projects

Solution-Phase Covalent Footprinting and Crosslinking

We are developing new methods using solution probes and native mass spectrometry to characterize protein structural dynamics. Specifically, we are investigating mass spectrometric approaches intrinsically disordered proteins and proteins with flexible regions, their various functional proteoforms (co- and post-translationally modified variants), as well as protein-protein complexes formed by disordered and flexible proteins, funded by the MIRA grant. We are especially interested in proteoforms relevant in human disease and healthy cellular function.

Gas-Phase Covalent Footprinting

Covalent reactions in the gas phase are conducted using ion/ion chemistry, where the analyte is ionized by electrospray in one polarity while the reagent is ionized in the opposite polarity. Opposites attract and electrostatically reactive groups like sulfonates form gas-phase salt bridges with positively charged sites, speeding up reaction kinetics by orders of magnitude over bulk solution. We determine possible protonation sites and modification sites by electron dissociation techniques, allowing us to map the through-space distance between the electrostatically-sticky portion of the reagent and the covalent modification site to produce structural constraints. Currently, we are investigating the energetics of these reactions relative to the gas-phase energy landscape of model proteins, applying these reactions to measure structural changes in proteins induced by changes in solution conditions, and designing and testing new gas-phase covalent reagents.

Gas-Phase Electrostatic Reactions

  Electrostatic ion/ion reactions specifically target charged residues in the gas phase. They have the advantage of virtually no energy requirement and occur spontaneously under gentle conditions. We are applying these reactions to footprint the accessible charged residues and to determine structural constraints between charged residues in the gas-phase. We are currently developing different-length electrostatic linkers to increase the precision of our measurements. Our gas-phase structural methods and investigations are funded by an NSF CAREER Award.