The Mechanism of Protein Stabilization by Additives under Electrothermal Supercharging Conditions.
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Formation of highly charged protein ions from ammonium bicarbonate solution is a fascinating phenomenon referred to as “electrothermal supercharging”. Electrothermal supercharging by ammonium bicarbonate is proposed to be due to native protein destabilization in heated ESI droplets under high spray voltages in the presence of bicarbonate anion. Here, the effects of several additives on protein charge state distribution under electrothermal supercharging conditions were investigated by the addition of several small molecule additives. The changes in the observed charge state distributions (CSD) were measured by the ratios between the intensities of highest intensity charge states (HICS) of native and unfolded protein envelopes and shifts in lowest observed charge state (LOCS) and highest observed charge state (HOCS). This study demonstrated that temperature is the more consequential parameter compared to spray voltage in supercharging by ammonium bicarbonate, especially when using a nebulized micro-electrospray ionization source. Moreover, the effects of the amino acids on supercharging with ammonium bicarbonate were generally in good agreement with the extensive literature available on the stabilization or destabilization of proteins by amino acid additives. Serine and arginine showed no stabilization or a destabilizing effect, whereas the extent of protein supercharging was significantly reduced by proline and glycine, indicating a stabilizing effect. Confoundingly, hydroxyproline showed a destabilizing effect, despite reports that hydroxyproline is better at thermal stabilization of proteins than proline in bulk solution. Imidazole, on the other hand, provided the highest degree of stabilization against electrothermal supercharging, suggesting that the evaporative cooling process is more significantly capable of reducing electrothermal supercharging.
Panth, Rajendra, "The Mechanism of Protein Stabilization by Additives under Electrothermal Supercharging Conditions." (2022). Honors Theses. 3614.