On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry

Konermann, Lars; Liu, Zeyuan; Haidar, Yousef; Willans, Mathew J.; Bainbridge, Nicholas A.

doi:10.1021/acs.analchem.3c02546

Cited by 13 publications

(14 citation statements)

References 99 publications

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“…We anticipated that the spray solution would start near pH 6 or 10 and experience some level of buffering around pH 4.75 or 9.25 prior to ESI droplet desolvation. Increased buffer capacity is expected to maintain the droplet pH around the p K a of either acetate or ammonium during ESI, , which in turn is expected to enable more confident prediction of the pH surrounding the analyte during in-ESI HDX. This allows for characterization of how ESI affects labeling during in-ESI HDX.…”

Section: Resultsmentioning

confidence: 99%

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

Hatvany,

Liyanage,

Gallagher

2024

J. Am. Soc. Mass Spectrom.

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Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray hydrogen/deuterium exchange mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid-and basecatalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes.

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Section: Resultsmentioning

confidence: 99%

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

Hatvany,

Liyanage,

Gallagher

2024

J. Am. Soc. Mass Spectrom.

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“…In addition to the redox reactions mentioned above, ESI droplet size reduction as a result of evaporation and fission can contribute to pH fluctuations as ions such as NH 4 + or H + become increasingly concentrated and acidify protein-containing droplets until the release of analyte ions. , Due to the faster evaporation of ammonia relative to acetic acid, droplets continue to acidify during their lifetime until the droplet pH stabilizes around the p K a of acetic acid, regardless of the initial [AmAc]. , Bal and co-workers highlighted that the lack of AmAc’s buffering capacity at neutral pH leads to significant sample acidification during ESI and dissociation of high-affinity Cu(II)-peptide complexes during native ESI-MS analysis . Bicarbonate salts (p K a 6.4) have proven to cause protein unfolding due to CO 2 gas evolution and Hofmeister effects, leading to disrupted protein–ligand interactions. − Identifying volatile and ESI-compatible additives that provide buffering capacity at physiological pH is crucial as currently there is no viable buffer available for neutral pH conditions.…”

Section: Introductionmentioning

confidence: 99%

“…+ or H + become increasingly concentrated and acidify protein-containing droplets until the release of analyte ions. 23,27 Due to the faster evaporation of ammonia relative to acetic acid, droplets continue to acidify during their lifetime until the droplet pH stabilizes around the pK a of acetic acid, regardless of the initial [AmAc]. 23,27 Bal and co-workers highlighted that the lack of AmAc's buffering capacity at neutral pH leads to significant sample acidification during ESI and dissociation of high-affinity Cu(II)-peptide complexes during native ESI-MS analysis.…”

Section: ■ Introductionmentioning

confidence: 99%

“…23,27 Due to the faster evaporation of ammonia relative to acetic acid, droplets continue to acidify during their lifetime until the droplet pH stabilizes around the pK a of acetic acid, regardless of the initial [AmAc]. 23,27 Bal and co-workers highlighted that the lack of AmAc's buffering capacity at neutral pH leads to significant sample acidification during ESI and dissociation of high-affinity Cu(II)-peptide complexes during native ESI-MS analysis. 21 Bicarbonate salts (pK a 6.4) have proven to cause protein unfolding due to CO 2 gas evolution and Hofmeister effects, leading to disrupted protein−ligand interactions.…”

Section: ■ Introductionmentioning

confidence: 99%

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Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry

Davis,

Velyvis,

Vahidi

2023

Anal. Chem.

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Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant "buffer" used, ammonium acetate (AmAc) with pK a values of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0−7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pHsensitive interactions. Consequently, the discovery of volatile, ESIcompatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pK a values of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome c and myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein−ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.

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“…Ammonium acetate and ammonium bicarbonate are common volatile mobile phase salts used with ESI detection. [12][13][14][15][16][17] interactions which cause ion-suppression or remain in the instrument after use. 18 Ammonium fluoride (NH 4 F) has been reported in the literature across applications as an effective volatile mobile phase additive in improving ESI sensitivity and is hypothesized to work as a sequestration agent.…”

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confidence: 99%

Assessment of ammonium fluoride as a mobile phase additive for sensitivity gains in electrospray ionization

McFadden,

Ames

2023

Analytical Science Advances

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Ammonium fluoride has been shown to improve sensitivity when using electrospray ionization (ESI) coupled with mass spectrometry (MS). Recent internal investigation furthered that claim, through the observation of improved sensitivity when analyzing steroid molecules. This work focuses on extending those observations to other small molecules to understand the impact ammonium fluoride has on detection sensitivity with optimized instrument conditions. Using conventional liquid chromatography ESI‐MS we investigated sensitivity differences between ammonium fluoride, formic acid, or ammonium hydroxide as mobile phase additives. Full source optimization was performed for nine compounds at three different organic concentrations (30%, 60%, or 90%) with formic acid, ammonium fluoride, and ammonium hydroxide adjustment. Optimization results were compiled to generate individual methods by compound, polarity, mobile phase, and organic concentration. Flow injection analysis was performed with fully optimized methods to compare compounds across different solvent systems under optimal conditions. Negative ESI data showed 2–22‐fold sensitivity improvements for all compounds with ammonium fluoride. Positive ESI data showed > 1–11‐fold improvement in sensitivity for four of seven compounds and no change for three of seven compounds with ammonium fluoride. Ammonium fluoride improved ESI− sensitivity for all compounds studied when using optimized source conditions. Investigation with ESI+ analyses showed mixed results, with four of seven compounds showing improvement and others showing equivalency or slight loss in sensitivity, suggesting potential sensitivity gains for some analogs with ESI+.

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On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry

Cited by 13 publications

References 99 publications

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides

Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry

Assessment of ammonium fluoride as a mobile phase additive for sensitivity gains in electrospray ionization

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