Over more than a decade ion mobility spectrometry (IMS) has been employed to great effect in the field of native mass spectrometry-based structural biology. In this field IMS has been used for separating protein oligomeric states, co-populated conformations and for measuring experimental collision cross-sections for use in determining protein complex architecture. In addition, from an early stage the potential of pre-IMS activation in probing gas phase protein unfolding pathways and stabilities was pursued. This experiment, called collision-induced unfolding (CIU) has spawned a whole field of research into probing protein domain architecture, protein-ligand stabilization and therapeutic antibody comparisons. Here we describe an IMS-based instrument platform with IMSn functionality which allows novel protein CIU studies to be performed in which protein sub-populations can be mobility-selected for further rounds of IMS. Furthermore, by adding an activation step in between IMS experiments, sequential rounds of unfolding can be performed on the same ion populations allowing greater insight into protein unfolding pathways.
Human TTR (Sigma Aldrich) was prepared at a concentration of 4 micromolar in 200 mM ammonium acetate. Native ion mobility experiments were performed on a cyclic ion mobility-enabled Q-Tof (ESI-Q-cIM-Tof) system fitted with an extended time-of-flight mass analyser, a segmented quadrupole transfer ion guide and dual gain ADC. In addition to unfolding studies the ability to mobility-select after quadrupole isolation and activation allows high mobility-resolution interrogation of ligands released from protein-ligand complexes.