Abstract
Thermal denaturation of holomyoglobin (hMb) in solution (10 mM ammonium acetate at pH = 4.5, 6.8, and 9.0) was monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques to characterize the stability and investigate structural changes involved in unfolding. We utilize two experimental approaches to induce thermal denaturation: a variable-temperature electrospray ionization (vT-ESI) source that heats the bulk solution in the ESI emitter, and a variable-power 10.6 μm CO(2) laser that rapidly heats nanodroplets produced by ESI. These two approaches sample different time scales of the denaturation process; long time scales (seconds to minutes) where the system is at equilibrium using the vT-ESI approach and shorter time scales (μs) by rapid droplet heating in which the system is in a pre-equilibrium state. Increasing the solution temperature (from 28 to 95 °C in the vT-ESI experiments) shifts the charge state distribution from low charge states ([M + 7H](7+) to [M + 9H](9+)) to more highly charged species. This is accompanied by loss of the heme group to yield the apomyoglobin (aMb) species, indicating that the protein has unfolded. Monitoring the formation of aMb and the shift in average charge states of aMb and hMb with solution temperature allows for relative quantitation of their individual stabilities, highlighting the stabilizing effects of heme binding. We compare the degree of unfolding induced by heating the bulk solution (using vT-ESI) to the laser droplet heating approach and find that the rapid nature of the laser heating approach allows for transient pre-equilibrium states to be sampled.