Session: Parallel session 2 - Ion mobility spectrometry

New insights about collision induced unfolding, future uses, and what is actually happening ?

Collision induced unfolding (CIU) is an experimental workflow based on the use of ion mobility – mass spectrometry (IM-MS). In brief, CIU experiments rely on the vibrational excitation of the ions by ion-neutral collisions which are performed before the ion mobility cell at defined acceleration voltages while monitoring the arrival time distribution (ATD) or the collision cross section (CCS). The results are mapped as CIU heatmaps plotting the ATD or CCS values against the activation energy at selected charge states of biopolymers, e.g. intact proteins. CIU heatmaps probe the energy landscapes due to the higher order structures (HOS) in vacuum of the ions under slow heating activation. Such heatmaps could be used to discriminate e.g. intact homologuous proteins or isoforms for biophysics, quality control or discrimination of biosimilars. The energy axis of the CIU heatmaps is provided in the laboratory energy frame because the frequency of collisions and the energy transferred per collisional event are unknown and are methods and instruments dependent.



We propose the use of thermometer ions, based on benzylpyridinium ions, model peptides (e.g. leucine enkephaline) and proteins (e.g. cytochrome C) to normalize the CCS and energy axes in terms of energy transferred to the ions during CIU experiments under different collisional regime, i.e. collisional events against He or Ar, at different pressures, and various activation energy provided by the Trap CE, Trap bias, or He DC acceleration voltages in a traveling wave IM-MS instrument (Waters Synapt G2Si). CIU heatmap obtained from different IM-MS instruments is under investigation to determine how well the CIU heatmaps can be transferred from instruments to instruments under closely related experimental conditions. Additionally our results support that CIU is not only driven by motion of protein domains or disorganization of their HOS but also because of proton transfer events from residues and backbone proton acceptors.