Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

Abstract

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that continues to grow in utilization due to its inherently high resolution and reduced instrumental footprint. In currently deployed instruments, however, typical modes of collisional activation do not precede IMS analysis and thus, the instruments are incapable of performing CIU workflows. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, β-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.

Characterizing the Top-down Sequencing of Protein Ions Prior to Mobility Separation in a timsTOF

“…Incrementally increasing the Δ6 voltage above this value gradually recruits more charge states to dissociate (Figure 2A, S4A, and S5A). As shown in our previous manuscripts, 17,33 increased tunnel-in pressure has a negative impact on dissociation.…”

“…We further characterized the relationship of tunnel-in pressure and Δ6 by examining the collision-induced unfolding of protein ions and found that higher pressures limited the maximum internal energy and degree of protein conformational isomerization. 33…”

“…While robust dissociation and product ion generation is possible at 1.5 mbar, our recent results have found that ion internal energies are inversely correlated with tunnel-in pressure. 17,33 To systematically examine how this relationship influences the dissociation of protein ions and the generation of sequence coverage across a series of protein ion masses, we directly infused ubiquitin, CytC, and βLG and compared how steadily increasing Δ6 values influences precursor and product ion abundances at tunnel-in pressures of 1.35, 1.5, 1.75, and 2.0 mbar. As in 1.5 mbar, no dissociation is observable in any protein or pressure when Δ6= 30 V (Figure 2A, S4A, and S5A).…”

Characterizing the Top-down Sequencing of Protein Ions Prior to Mobility Separation in a timsTOF

“…Incrementally increasing the Δ6 voltage above this value at 1.5 mbar gradually recruits more ubiquitin charge states to dissociate until five charge state are fully depleted at 150 V (Figure 2A). As demonstrated in our previous manuscripts, 17,33 increased tunnel-in pressure has an inverse relationship with ion activation and only the 14+ and 13+ charge states of ubiquitin dissociate at 2 mbar even when the highest value of Δ6 is applied (Figure 2A). Similar trends in ion dissociation are observed for CytC, and βLG as they are subjected to Δ6 activation at various tunnel-in pressures (Figure S4A and S5A).…”

Application of Multiple Length Cross-linkers to the Characterization of Gaseous Protein Structure

“…Indeed, increasing the temperature (i.e., through collisional activation) of compact cytochrome c structures in the gas phase does produce extended states that compact down to the original CCS values, with the compact structures displaying a "permanent" increase in CCS. [58][59] With these considerations, the most accurate modeling of gaseous structures of well-folded proteins would need to keep temperatures low enough that the gas phase rearrangement barriers are not reached. Additionally, using the crystal structure of folded proteins, followed by relaxation in the gas phase, accurately reproduces experimental observables.…”