title: Ultrahigh-resolution differential ion mobility separations of conformers for proteins above 10 kDa: onset of dipole alignment?
abstract: Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential or field asymmetric waveform IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with the same peak width and resolving power up to ∼400 as for peptides. The present work explores the reach of this approach for larger proteins, exemplified by cytochrome c and myoglobin. Resolution similar to that for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of the present technique to proteins under ∼20 kDa. This capability may enable one to distinguish whole proteins with differing residue sequences or localizations of post-translational modifications. Small features at negative compensation voltages that markedly grow from cytochrome c to myoglobin indicate the dipole alignment of rare conformers in accord with theory, further supporting the concept of pendular macroions in FAIMS.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory , P.O. Box 999, Richland, Washington 99352, United States.
authors.forename: Alexandre A
authors.initials: AA
authors.lastname: Shvartsburg
mesh_terms: D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D009211:Myoglobin / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D014907:Whales
chemical_list: D009211:Myoglobin; D045304:Cytochromes c