posted on 2023-12-11, 14:33authored byRoshan Javanshad, Rajendra Panth, Andre R Venter
The surprising formation of highly charged protein ions
from aqueous
ammonium bicarbonate solution is a fascinating phenomenon referred
to as electrothermal supercharging (ETS). Although the precise mechanism
involved is not clearly understood, previous studies predominantly
suggest that ETS is due to native protein destabilization in the presence
of bicarbonate anion inside the electrospray ionization droplets under
high temperatures and spray voltages. To evaluate existing hypotheses
surrounding the underlying mechanism of ETS, the effects of several
additives on protein charging under ETS conditions were investigated.
The changes in the protein charge state distributions were compared
by measuring the ratios between the intensities of highest
intensity charge states of native and unfolded protein envelopes
and shifts in the lowest and highest observed charge states. This study demonstrated that source temperature plays a more important
role in ETS compared to spray voltage, especially when using a nebulized
microelectrospray ionization source. Moreover, the effect of amino
acids on ETS were generally in good agreement with the extensive literature
available on the stabilization or destabilization of proteins by these
additives in bulk solution. Among the natural amino acids, protein
supercharging was significantly reduced by proline and glycine; however,
imidazole provided the highest degree of noncovalent complex stabilization
against ETS, outperforming the amino acids. Overall, our study shows
that the simple addition of stabilizing reagents such as proline and
imidazole can reduce the extent of apparent protein unfolding and
supercharging in ammonium bicarbonate solution and provide evidence
against the roles of charge depletion and thermal unfolding during
ETS.