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.
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In protein ESI-MS with holomyoglobin under native conditions, high charge states formed and their signal intensities increased with buffer ionic strength.
“As illustrated in Figure 6, high charge states (as in denaturing conditions) were formed in native conditions and their signal intensities increased proportionally to the increase in buffer ionic strength.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [101]
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- Authors
- Hedges, J. B., Vahidi, S., Yue, X., & Konermann, L.
- Affiliations
- Western University (CA)
- Corresponding author
- Konermann, Lars
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2013-06-18
- Volume
- 85
- Issue
- 13
- Pages
- 6469-6476
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W2003162850
- Cited by
- 109
- References
- 76
- Citations by year
- 2026: 15 · 2025: 10 · 2024: 6 · 2023: 15 · 2022: 11 · 2021: 10 · 2020: 8 · 2019: 9 · 2018: 3 · 2017: 4 · 2016: 5 · 2015: 5 · 2014: 7 · 2013: 1
- FWCI
- 2.69
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Canada Research Chairs
- Natural Sciences and Engineering Research Council of Canada
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Engineering › Electrical and Electronic Engineering › Electrohydrodynamics and Fluid Dynamics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- ammonium bicarbonate, ammonium acetate, electrospray ionization, protein unfolding, foaming, CO2 degassing, droplet heating, collisional heating, high charge states
- MeSH terms
- Animals, Bicarbonates, Gases, Horses, Myoglobin, Spectrometry, Mass, Electrospray Ionization, Protein Unfolding
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