Addressing a Common Misconception: Ammonium Acetate as Neutral pH “Buffer” for Native Electrospray Mass Spectrometry
Abstract
Native ESI-MS involves the transfer of intact proteins and biomolecular complexes from solution into the gas phase. One potential pitfall is the occurrence of pH-induced changes that can affect the analyte while it is still surrounded by solvent. Most native ESI-MS studies employ neutral aqueous ammonium acetate solutions. It is a widely perpetuated misconception that ammonium acetate buffers the analyte solution at neutral pH. By definition, a buffer consists of a weak acid and its conjugate weak base. The buffering range covers the weak acid pK a ± 1 pH unit. NH 4 + and CH 3 -COO - are not a conjugate acid/base pair, which means that they do not constitute a buffer at pH 7. Dissolution of ammonium acetate salt in water results in pH 7, but this pH is highly labile. Ammonium acetate does provide buffering around pH 4.75 (the pK a of acetic acid) and around pH 9.25 (the pK a of ammonium). This implies that neutral ammonium acetate solutions electrosprayed in positive ion mode will likely undergo acidification down to pH 4.75 ± 1 in the ESI plume. Ammonium acetate nonetheless remains a useful additive for native ESI-MS. It is a volatile electrolyte that can mimic the solvation properties experienced by proteins under physiological conditions. Also, a drop from pH 7 to around pH 4.75 is less dramatic than the acidification that would take place in pure water. It is hoped that the habit of referring to pH 7 solutions as ammonium acetate "buffer" will disappear from the literatur…
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In the presence of ammonium acetate, the pH may drop to values as low as 4.75 ± 1 in the final ESI droplets, reflecting the pKa of acetate buffer.
“In the presence of ammonium acetate, the pH may drop to values as low as 4.75 ± 1 in the final ESI droplets, reflecting the pKa of acetate buffer50.”
From Native metabolomics identifies the rivulariapeptolide family of protease inhibitors (2022) · cites this paper as [50]
CZE separation has frequently been used to analyze glycosylated peptides and glycans, and CZE via TDMS has been used for glycosylation characterization.
“CZE separation has frequently been used to analyze glycosylated peptides and glycans49,57,80,81 and CZE separation via TDMS was used for glycosylation characterization82.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [81]
Ammonium acetate does not form a buffer system that can effectively maintain macromolecular complexes in solution at physiological pH.
“Since ammonium acetate does not constitute a buffer system that can effectively maintain macromolecular complexes in solution at physiological pH conditions81,100”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [81]
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- Authors
- Konermann, L.
- Affiliations
- Western University (CA)
- Corresponding author
- Konermann, Lars
- ORCID
- Konermann, Lars
- Venue
- Journal of the American Society for Mass Spectrometry
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-07-14
- Volume
- 28
- Issue
- 9
- Pages
- 1827-1835
- ISSN
- 1044-0305 (ISSN-L)
- OpenAlex
- W2735371049
- Cited by
- 243
- References
- 96
- Citations by year
- 2026: 22 · 2025: 26 · 2024: 39 · 2023: 30 · 2022: 28 · 2021: 28 · 2020: 33 · 2019: 27 · 2018: 9 · 2017: 1
- FWCI
- 9.46
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of the American Society for Mass Spectrometry (publishedVersion)
- PubMed (publishedVersion)
- Scholarship@Western (Western University) (submittedVersion)
- Funders
- 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 acetate, pH buffering, conjugate acid-base pairs, pKa, protein solvation, positive ion mode
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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