Separation of Protein Conformers by Differential Ion Mobility in Hydrogen-Rich Gases

Proteins in solution or the gas phase tend to exhibit multiple conformational families, each comprising distinct structures. Separation methods have generally failed to resolve these, with their convolution producing wide peaks. Here, we report full separation of > 10 conformers for most ubiquitin charge states by the new approach of differential ion mobility spectrometry (field asymmetric waveform ion mobility spectrometry, FAIMS) employing H-2/N-2 gas mixtures with up to 85% H-2. The resolving power (up to 400) is five times the highest previously achieved (using He/N-2 buffers), greatly increasing the separation specificity. The peak widths match the narrowest obtained by FAIMS for any species under the same conditions and scale with the protein charge state (z) and ion residence time (t) as z(-1/2) and t(-1/2), as prescribed for instrumental (diffusional) broadening. This suggests resolution of specific geometries rather than broader ensembles.