The Effect of Amino Acids on Protein Stability During Electrothermal Supercharging

In a new study in the Journal of the American Society for Mass Spectrometry, scientists from Western Michigan University tested how amino acids can affect protein stability during electrothermal supercharging (ETS) (1).

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ETS refers to the formation of highly charged protein ions stemming from aqueous ammonium bicarbonate solution. The mechanisms behind this process are not fully understood, though studies do suggest that ETS occurs to native protein destabilization in electrospray ionization droplets that are in the presence of bicarbonate anions, under high temperatures and spray voltages.

To evaluate existing hypotheses, the team investigated the effects of different additives on protein charging under ETS conditions. Changes in protein charge state distributions were compared by measuring the ratios between the intensities of the highest intensity charge states of native and unfolded protein envelops, in addition to shifts in the lowest and highest observed charge states.

Following the experiment, the scientists deduced that source temperature plays an important role in ETS compared to spray voltage, especially when a nebulized microelectrospray ionization source is used. Amino acids’ effect on ETS seemed to agree with prior literature about the stabilization or destabilization of proteins by these additives. Protein supercharging was minimized by proline and glycine, with imidazole allowing for the most noncovalent complex stabilization against ETS, compared to the other amino acids. The study’s findings showed that adding stabilizing reagents, such as proline and imidazole, can reduce protein unfolding and supercharging in ammonium bicarbonate solution, providing evidence against how charge depletion and thermal unfolding can affect ETS.

Reference

(1) Javanshad, R.; Panth, R.; Venter, A. R. Effects of Amino Acid Additives on Protein Stability during Electrothermal Supercharging in ESI-MS. J. Am. Soc. Mass Spectrom. 2024, 35 (1), 151–157. DOI: https://doi.org/10.1021/jasms.3c00377