Gas Phase Stability of Protein Ions in a Cyclic Ion Mobility Spectrometry Traveling Wave Device

Abstract

Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multipass traveling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed, which were used to obtain more detailed collision-induced unfolding pathways for Cytochrome C. The instrument geometry provides unique capabilities with the potential to expand the field of protein analysis via IM-MS.

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation

“…6). While there is a general correlation between the number of unfolding steps and the number of domains in a protein, we note that is charge-state dependent phenomenon 15 , and that multi-step unfolding of various single-domain proteins has been reported [47][48] , revealing a richness of information in the CIU trajectory beyond domain stoichiometry.…”

“…In line with Occam's razor, we used the simplest model afforded by the data and, though the model produced seemingly sensible quantitative insights into the data, it is possible (indeed, likely) that there are complexities to the mechanism of SOD1 unfolding and dissociation to which our data is not sensitive, and hence we are unjustified in modelling. In this regard, recent developments in IM-MS technology that allow specific conformers can be activated individually such that specific dissociation channels can be interrogated hold great promise in increasing the information content of the gas-phase activation experiment 47 . Such experiments will add to the arsenal of experimental and analysis approaches that will ultimately combine to allow the exhaustive, quantitative dissection of native proteins through all levels of their structure, from non-covalent assembly to peptide fragments.…”

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology

“…How this phenomenon manifests itself for proteins of all sizes and shapes, and for other types of macromolecules, is currently not known, but neglecting these effects is unlikely to be the major source of bias; more important perhaps are the perturbations the charges make to the structure (see below). Nevertheless, given the increasingly sophisticated questions that IM-MS is being used to answer, and the higher performance IM-MS instruments that have become available [33][34] , considerable scope remains to ensure that local charges and interaction potentials are effectively accommodated in CCS calculations.…”

Application of a novel cyclic ion mobility‐mass spectrometer to the analysis of synthetic polymers: A preliminary evaluation

“…This arrangement is capable of manipulating the ions to effect injection and ejection into and out of the ion mobility separator, enabling the use of complex ion mobility sequences. A full description of cyclic sequence methods, and how they are developed, is given elsewhere; in addition, full details of the timings used for the methods employed within this work are presented in Table S1 (supporting information). These sequences were developed in a stepwise manner while observing the arrival times of key ions of interest to track the number of passes of the cyclic device completed by the ions of interest.…”

“…Early work on this concept was carried out by Merenbloom and co‐workers, while the commercial realisation of this approach has recently been completed by Giles and co‐workers to deliver an integrated cyclic ion mobility‐mass spectrometry (cIM‐MS) system. The most obvious technical advancement from this type of instrument is the potential for improved IM resolution; however, novel developments in ion manipulation provide further scope for new experimental approaches and improvements to current IM acquisitions . The work presented here explores both the effects of increased IM resolution and the possibilities for new experimental designs for synthetic polymer analysis.…”