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

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 multi-pass travelling 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 novel instrument geometry provide unique capabilities with the potential to expand the field of protein analysis via IM-MS.</div

Cyclic Ion Mobility – Collision Activation Experiments Elucidate Protein Behaviour in the Gas-Phase

Elucidating the properties of intrinsically disordered proteins (IDPs) and unfolded and partially folded states of globular proteins is challenging owing to their heterogeneous and dynamic nature. Protein unfolding and misfolding is a key feature of a broad range of debilitating diseases, whilst the conformational propensities of intrinsically disordered proteins can play a significant role in modulating their activity, and the properties of unfolded states of globular proteins modulates their stability and tendency to aggregate. Ion mobility-mass spectrometry (IM-MS) is a powerful method for interrogating these systems, however limits in resolution and the difficulty in probing the energetics of interconversions amongst heterogeneous ensembles are major issues. Herein, using a quadrupole/cyclic-IM/ time-of-flight MS instrument, we show how the combination of precursor mass selection, mobility selection (IMn) and collisional activation (CA) allows the elucidation of complicated gas-phase dynamic behavior. The methodology employed is general and is demonstrated using a classic model globular protein, cytochrome C, and an aggregation-prone IDP, amylin. CA allows investigations of protein conformational dynamics and unfolding in the gas-phase for heterogeneous mixtures, whilst the additional precursor mass selection capability provides high resolution and selectivity, facilitating more in-depth investigation. Understanding protein dynamics in the gas-phase will allow greater insight into protein behaviour and allow application of gas-phase techniques to clinically relevant systems. </i