Mass spectrometry (MS) has developed into a powerful tool for investigating the structure of proteins. The advent of electrospray ionization MS (ESI-MS) made it possible to transmit large, non-covalent complexes without significantly disrupting their structure. When ESI-MS is coupled with ion mobility spectrometry (IMS) it becomes possible to investigate the conformational dynamics of a biomolecule. Furthermore, a temperature-controlled ESI-MS (TC-ESI-MS) source can be used to allow for the continuous monitoring of temperature-induced changes in a biomolecule, providing insight into its thermodynamics and aggregation properties. For larger complexes, this information cannot be readily obtained using conventional methods, highlighting the usefulness of this approach. However, this analysis requires careful instrument tuning to ensure that a biomolecule is transmitted without perturbing its gas-phase structure. The goal of this work was to provide a framework for tuning a cyclic IMS mass spectrometer for the analysis of biomolecules and demonstrate how this instrument can be coupled with TC-ESI-MS to elucidate the structure of temperature-induced mDa protein aggregates.

The cyclic IM mass spectrometer used in this work has three phases during standard IMS experiments: an ‘inject’ phase, where ions are moved on to the cyclic array located in the cyclic IMS cell, a ‘separate’ phase, where the ions a pushed off the array for IMS separation, then finally, the ‘eject and acquire’ phase in which ions pass around the cyclic IMS cell, and are guided out of the system by the cyclic array. The two settings which had the greatest effect on ion transmission were the array offset and the so-called "racetrack bias" during the ‘separate’ phase. We found that similar voltages are required for effective ion transmission, otherwise, ions get caught in the array, and will not move around the cyclic IMS cell, which is evident in the IMS chromatogram as a peak with a drift time starting just after the ‘separate’ phase. The degree of these effects was dependent on the mass of the protein being analyzed. Optimized IMS settings were then used in the TC-ESI-MS analysis of Jack bean urease. This analysis was performed to investigate temperature-induced effects on the structure of this protein. Prior to heating, the two highest oligomeric states of this protein were the hexamer (~ 550 kDa, native) and a dodecamer (1.1 MDa, nonspecific). An increase in temperature led to urease forming octadecamers. Three distinct gas-phase conformations were observed for the dodecamers, the prevalence for two of which were affected by temperature.