Laser-Induced Denaturation of Cytochrome <i>c</i> in Electrospray Droplets

Structural transitions of the model system cytochrome <i>c</i> (Cyt <i>c</i>) were monitored by ion mobility spectrometry (IMS) and mass spectrometry (MS) paired with two methods to heat proteins: a variable-temperature electrospray ionization (vT-ESI) source to heat the bulk protein solution and a 10.6 μm CO₂ laser to rapidly heat ESI droplets containing the protein. Previous evidence from our group suggests that information about time-dependent protein structural transitions can be accessed by irradiating protein droplets of different sizes. In this paper, a new method to control droplet sizes is introduced where the distance between the ESI emitter and laser path is altered to produce larger or smaller droplets, yielding a simple and robust means of accessing different protein unfolding timescales. Herein, increasing the temperature of a solution of Cyt <i>c</i> in water at pH 4 via vT-ESI (from 27 to 80 °C) shifts the distribution of states from a relatively folded ensemble consisting of low charge states to a distribution of elongated structures that are observed as highly charged species. Rapid heating of ESI droplets (containing Cyt <i>c</i>) with a variable-power CO₂ laser yields a similar shift in the mass spectra with increasing laser power. To investigate the conformational changes accessible within the lifetime of the heated droplets, four different tip sizes as well as several different distances between the ESI emitter and laser path are studied. Slight changes in droplet size can greatly alter the response of the protein to the laser field. The maximum observable charge state upon laser heating appears to be limited by the size of the ESI droplet prior to entering the laser field. The dependence of these distributions on droplets sizes leads us to propose that laser-induced denaturation in ESI droplets is stopped before an equilibrium distribution of conformers can be reached─providing a means of kinetically trapping ensembles of states. Therefore, we provide a simple correlation between droplet size, percent protein folded, and appropriate experimental distance to suggest a framework for robust studies of protein denaturation in ESI droplets.