Optimized Low-Field Differential Ion Mobility Separations with High-Resolution Mass Spectrometry for Top-Down Proteomics

Ion mobility spectrometry (IMS) with mass spectrometry (MS) is a versatile approach to simplify mixtures, distinguish isomers, and elucidate molecular geometries. While linear IMS relies on the mobility <i>K</i> at moderate normalized electric field <i>E</i>/<i>N</i>, field asymmetric waveform IMS (FAIMS) captures its increment Δ<i>K</i> at high <i>E</i>/<i>N</i> levels causing ion heating. A novel nonlinear low-field differential (LOD) IMS separates large macroions, leveraging their dipole alignment. The IMS/MS mass limit is set by and advances with the MS stages. As the ever-heavier macromolecules and more complex samples demand ultimate MS resolution and accuracy, coupling IMS to Fourier-Transform (FT) MS has become essential in proteomics and structural biology. Here, we integrate LODIMS using the bisinusoidal (bis) or augmented flexible rectangular waveforms with Orbitrap MS and MS/MS and employ this platform to explore Concanavalin A (ConA, 25.6 kDa) comprising the natural noncovalent forms. The isotopic envelopes for all pieces were disentangled by MS. The intact ConA strongly aligns for most charge states, trivially separating from the (rotary) pieces for improved detection of both. The rectangular and scaled bis waveforms yield consistent directional cross sections and dipole moments─duly smaller than for the larger proteins. The newly identified peptides uncover an endogenous cleavage at N¹⁶² (plus the known N¹¹⁸) and thus two additional ConA forms. This work demonstrates the capability of LODIMS to align smaller proteins, reliably assess their directional cross sections and (weaker) dipoles, and facilitate top-down proteomics with the discovery of novel proteoforms.