Directional Ion Mobility Spectrometry of Dipole-Aligned Macromolecules: a New Toolbox for Structural Biology and Top-Down Proteomics

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Alexandre Shvartsburg at Wichita State University is developing a novel toolbox for the separation and characterization of macromolecules based on the gas-phase mobilities of aligned ions. For a century since the emergence of ion mobility spectrometry (IMS) around 1900, all methods dealt with freely rotating ions. However, most macroions are polar and can align in electric field as a compass aligns with the Earth magnetic field. The transfer of such pendular ions is governed by their projections in given directions, not the average over all orientations normally relevant to IMS. This enables more detailed identification and characterization of ion geometries, in parallel to the police files comprising the face and profile photos of suspects. Such studies further capture the dipole moments also related to the molecular structure. For complex mixture analyses and broad community exploration, these approaches will be coupled to ultrahigh-resolution mass spectrometry (MS) at a major national laboratory. This research is complemented by teaching new specialized courses at selected universities across the US. Essentially all macroions have permanent dipole moments. Above a certain threshold, a sufficient electric field overcomes the thermal rotation yielding pendular states with the mobility controlled by the directional cross section across the dipole (CCSDir). Then the field dependence of mobility reveals the previously unknown moments and CCSDir values, providing new structural information complementary to regular IMS and establishing novel macromolecular separations. While this paradigm was originally shown with the bisinusoidal waveforms from FAIMS, the slow macroion motion permits lower frequencies allowing practical rectangular waveforms. That capability would now be extended to weaker dipoles by raising the waveform amplitude and narrowing the gap for stronger field, with more flexible alignment achieved by varying the waveform profile and gas temperature. The helium buffers compatible with weak field would magnify the differential mobility and facilitate the connection to ion morphologies via first-principles modeling as with linear IMS. These digital waveforms accommodate an arbitrary number of flat segments, allowing us to implement and evaluate the long-standing concept of higher-order differential IMS based on the terms of mobility(field) function beyond quadratic. These and other unprecedented separations ensuing from the dynamic dipole locking would be integrated with Fourier Transform MS at National High Magnetic Field Laboratory, including the unique 21-Tesla Fourier Transform Ion Cyclotron Resonance system delivering ultimate MS resolution. The IMS/MS platforms and technologies resulting from this project would expand the frontiers of integrative structural biology and top-down proteomics. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.