Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td

Abstract

Since its inception in 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) has been implemented at or near ambient gas pressure. We recently developed FAIMS at 15−30 Torr with mass spectrometry and utilized it to analyze amino acids, isomeric peptides, and protein conformers. The separations broadly mirrored those at atmospheric pressure, save for larger proteins that (as predicted) exhibited dipole alignment at ambient but not low pressure. Here we reduce the pressure down to 4.7 Torr, allowing normalized electric fields up to 543 Tddouble the maximum in prior FAIMS or IMS studies of polyatomic ions. Despite the collisional heating to ∼1000 °C at the waveform peaks, the proteins of size from ubiquitin to albumin survived intact. The dissociation of macromolecules in FAIMS appears governed by the average ion temperature over the waveform cycle, unlike the isomerization controlled by the peak temperature. The global separation trends in this "superhot" regime extend those at moderately low pressures, with distinct conformers and no alignment as theorized. Although the scaling of the compensation voltage with the field fell below cubic at lower fields, the resolving power increased and the resolution of different proteins or charge states substantially improved.

Assessing the Dipole Moments and Directional Cross Sections of Proteins and Complexes by Differential Ion Mobility Spectrometry

“…For a given protein, the alignment increased at higher charge states (z for monomers, z d for dimers) that allow both higher partial charges and their greater separation upon unfolding driven by Coulomb repulsion. 35 No features at E C < 0 were duly found in LP-FAIMS 15 for proteins up to ∼100 kDa (with projected p < 5000 D). The E C scans for aligned ions depend on their dipole properties, but the extraction of those from data is complicated by confounding FAIMS effects.…”

“…Most systems run at or near ambient pressure (P) although low-pressure (LP) FAIMS at P ∼ 0.01− 0.02 atm was recently demonstrated. 15 A major advantage of FAIMS over linear IMS is the greater orthogonality to MS: the correlation between mass (m) and size (and thus K) for isomers and other compositionally similar species is intrinsically tight, 5,16 while that between m and ΔK is much looser (by 3× to 6× for lipids and peptides). 17−19 This allows FAIMS to outperform linear IMS of even higher resolving power (R) in separations of isomeric lipids, 20 variant modified peptides, 19,21 protein conformers, 22 isotopologues, 23 and isotopomers.…”

“…17−19 This allows FAIMS to outperform linear IMS of even higher resolving power (R) in separations of isomeric lipids, 20 variant modified peptides, 19,21 protein conformers, 22 isotopologues, 23 and isotopomers. 23,24 However, as a structural tool, FAIMS could not compete with IMS 25−30 any geometry 1,25,31 and (ii) collisional heating of ions inherent to FAIMS routinely isomerized the fragile native or other biochemically relevant conformations of macromolecules and their complexes 15,32 or even fragmented the analytes. 33 The K(E) dependences agglomerate multiple physical phenomena.…”

“…Typical protein ions generated by electrospray ionization (ESI) have K 0 ∼ 0.7−1 cm 2 /(Vs) in the N 2 gas, 27,30 which yields p crit ∼ 300 Debye (D) at P = 1 atm but ∼30 kD at 0.01 atm. 15,35 Most chemical species are permanent dipoles. The moments, defined as the (magnitude of partial charges) × (distance between them), tend to increase for larger molecules that can carry more charges and permit their greater separation.…”

Effects of the LC mobile phase in vacuum differential mobility spectrometry-mass spectrometry for the selective analysis of antidepressant drugs in human plasma

“…While most DMS applications were developed on systems at atmospheric pressure, recently, a prototype DMS operating at low pressure regime 6–40 mbar, using a planar-gap stage within the MS instrument envelope extending the normalized electric field up to 543 Td, was reported by Shvartsburg et al [ 22 , 23 ]. Transit time below 30 ms makes vDMS compatible with LC-MRM/MS acquisition.…”