Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry.

Polar molecules align in electric fields when the dipole energy (proportional to field intensity E x dipole moment p) exceeds the thermal rotational energy. Small molecules have low p and align only at inordinately high E or upon extreme cooling. Many biomacromolecules and ions are strong permanent dipoles that align at E achievable in gases and room temperature. The collision cross-sections of aligned ions with gas molecules generally differ from orientationally averaged quantities, affecting ion mobilities measured in ion mobility spectrometry (IMS). Field asymmetric waveform IMS (FAIMS) separates ions by the difference between mobilities at high and low E and hence can resolve and identify macroion conformers based on the mobility difference between pendular and free rotor states. The exceptional sensitivity of that difference to ion geometry and charge distribution holds the potential for a powerful method for separation and characterization of macromolecular species. Theory predicts that the pendular alignment of ions in gases at any E requires a minimum p that depends on the ion mobility, gas pressure, and temperature. At ambient conditions used in current FAIMS systems, p for realistic ions must exceed approximately 300-400 Debye. The dipole moments of proteins statistically increase with increasing mass, and such values are typical above approximately 30 kDa. As expected for the dipole-aligned regime, FAIMS analyses of protein ions and complexes of approximately 30-130 kDa show an order-of-magnitude expansion of separation space compared with smaller proteins and other ions.