Traveling-wave ion mobility-mass spectrometry reveals additional mechanistic details in the stabilization of protein complex ions through tuned salt additives

Abstract

Ion mobility–mass spectrometry is often applied to the structural elucidation of multiprotein assemblies in cases where X-ray crystallography or NMR experiments have proved challenging. Such applications are growing steadily as we continue to probe regions of the proteome that are less-accessible to such high-resolution structural biology tools. Since ion mobility measures protein structure in the absence of bulk solvent, strategies designed to more-broadly stabilize native-like protein structures in the gas-phase would greatly enable the application of such measurements to challenging structural targets. Recently, we have begun investigating the ability of salt-based solution additives that remain bound to protein ions in the gas-phase to stabilize native-like protein structures. These experiments, which utilize collision induced unfolding and collision induced dissociation in a tandem mass spectrometry mode to measure protein stability, seek to develop a rank-order similar to the Hofmeister series that categorizes the general ability of different anions and cations to stabilize gas-phase protein structure. Here, we study magnesium chloride as a potential stabilizing additive for protein structures in vacuo, and find that the addition of this salt to solutions prior to nano-electrospray ionization dramatically enhances multiprotein complex structural stability in the gas-phase. Based on these experiments, we also refine the physical mechanism of cation-based protein complex ion stabilization by tracking the unfolding transitions experienced by cation-bound complexes. Upon comparison with unbound proteins, we find strong evidence that stabilizing cations act to tether protein complex structure. We conclude by putting the results reported here in context, and by projecting the future applications of this method.

Native ion mobility mass spectrometry reveals that small organic acid fragments impart gas‐phase stability to carbonic anhydrase II

“…Furthermore, the combination of stabilising anions and cations as neutral salts was found to have an additive stabilisation effect. 20 To this end, we examined the effect that the addition of KCl to CA II might have on protein stability under our conditions. At a final concentration of 60 µM we were able to observe multiple potassium adducts, both with and without bound acetate.…”

“…18 Bound anions have been proposed to enhance protein stability due to dissociative cooling which removes rotational and vibrational energy from the gas phase protein. 20 Based on the observation that inhibition of CIU through anion binding is related to protein affinity, we were curious as to whether this affect may also occur with organic molecules such as those encountered in fragment-based drug discovery (FBDD) campaigns. Typically, FBDD workflows utilise sensitive biophysical techniques to identify organic fragments, as a starting point for hit optimisation.…”

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding

“…At least three less compact conformations in addition to the native conformation were observed at 0% DMSO for the 15+ charge state of avidin, which was similar to previously reported. [19][20] Higher charge states of the avidin tetramer unfolded at lower CV, which can again be attributed to increased Coulombic destabilization. Unfolding pathways for the avidin tetramer were broadly similar in the presence of 50% DMSO (Figure 4b).…”

“…[14][15][16] Previous studies mainly by the Ruotolo group have explored the effect of various cations and ions on the CID and CIU stabilities of model proteins. [17][18][19][20] However, to our knowledge, no systematic study on the effect of DMSO on protein CID or CIU behavior has yet been reported.…”

Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding

“…For example, CIU results have been used to record the gas‐phase folding landscape of ubiqutin ions over a range of charge states using tandem IM instrumentation, with collisional activation regions between IM stages . Additionally, CIU of protein complexes has measured the stability of salt‐adducted assemblies, been used to assess stability enhancements in pathogenic mutants, and differentiate conformationally selective kinase inhibitors . Most recently, IM‐MS and CIU data have been used to ascertain the selectivity and stability of bound lipids within the mechanosensitive channel of large conductance from Mycobacterium tuberculosis , as well as E. coli aquaporin and ammonia channels …”