Effects of Ammonium Bicarbonate on the Electrospray Mass Spectra of Proteins: Evidence for Bubble-Induced Unfolding

Abstract

Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.

The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

“…Supercharging with Cr(III) resembles electrothermal supercharging [42–45] in that both methods can add a greater number of protons than there are highly basic sites on the biomolecule. Electrothermal supercharging is believed to result from conformational changes and denaturation when a biomolecule is exposed to factors that include temperature, voltage, ionic strength, and the development of gas bubbles in solution.…”

“…Flick and Williams have also reported that lanthanum(III) chloride can supercharge proteins [41]. In addition, an electrothermal supercharging method has been developed that involves ESI from aqueous ammonium or sodium salt solutions, with the extent of protonation being dependent on solution conditions and on the ESI capillary temperature and spray potential [42–45]. An important factor is protein unfolding induced by gas bubbles arising from the heated salt solutions [45].…”

“…In addition, an electrothermal supercharging method has been developed that involves ESI from aqueous ammonium or sodium salt solutions, with the extent of protonation being dependent on solution conditions and on the ESI capillary temperature and spray potential [42–45]. An important factor is protein unfolding induced by gas bubbles arising from the heated salt solutions [45]. A unique feature of electrothermal supercharging is that the number of protons added to the protein is often greater than the number of highly basic sites; in contrast, organic supercharging reagents normally do not protonate beyond the number of highly basic sites on the protein or peptide.…”

What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

“…Its usage has expanded to encompass different phenomena, including the increased charging associated with adduction of trivalent lanthanum cations [44], and “electrothermally–induced” denaturation from native solutions [45], subsequently linked to CO 2 -outgassing from bicarbonate buffer [46]. Here, we restrain application of the term “supercharging” to analytes’ increased positive or negative charge associated with limited addition of certain agents to native or denaturing solutions, but, for native ESI, only when that increase is not accompanied by classic denaturation signatures.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…Consequently, MS compatible solutions, such as aqueous ammonium acetate, ammonium bicarbonate or ammonium formate, are commonly used for native MS applications, and buffer exchange must be stringent [60]. Ammonium acetate is typically utilized for native MS applications, as ammonium bicarbonate has been shown to generate high protein charge states (relatively unfolded ions) when analyzed at pH 7, but this can be regulated by changing the capillary voltage and source temperature [61][62][63]. It is important to note that at pH 7, ammonium acetate does not function as a buffer at all, but is effective around pH 4.75 and 9.25 (pKa values of the CH 3 COOH/CH 3 COO À and NH 4 + /NH 3 acid/ base pairs, respectively) [64].…”