Field asymmetric waveform ion mobility spectrometry studies of proteins: Dipole alignment in ion mobility spectrometry?

Approaches to separation and characterization of ions based on their mobilities in gases date back to the 1960s. Conventional ion mobility spectrometry (IMS) measures the absolute mobility, and field asymmetric waveform IMS (FAIMS) exploits the difference between mobilities at high and low electric fields. However, in all previous IMS and FAIMS experiments ions experienced an essentially free rotation; thus the separation was based on the orientationally averaged cross-sections Omega(avg) between ions and buffer gas molecules. Virtually all large ions are permanent electric dipoles that will be oriented by a sufficiently strong electric field. Under typical FAIMS conditions this will occur for dipole moments > 400 D, found for many macroions including most proteins above similar to 30 kDa. Mobilities of aligned dipoles depend on directional cross-sections Omega(dir) ( rather than Omega(avg)), which should have a major effect on FAIMS separation parameters. Here we report the FAIMS behavior of electrospray-ionization-generated ions for 10 proteins up to similar to 70 kDa. Those above 29 kDa exhibit a strong increase of mobility at high field, which is consistent with predicted ion dipole alignment. This effect expands the useful FAIMS separation power by an order of magnitude, allowing separation of up to similar to 10(2) distinct protein conformers and potentially revealing information about Omega(dir) and ion dipole moment that is of utility for structural characterization. Possible approaches to extending dipole alignment to smaller ions are discussed.