Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry
Bradley T V Davis1, Algirdas Velyvis1, Siavash Vahidi1
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Analytical Chemistry
|November 24, 2023
Summary
New volatile buffers, 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA), maintain physiological pH for native ESI-MS. These buffers preserve macromolecular complexes, overcoming limitations of ammonium acetate for studying pH-sensitive interactions.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Mass Spectrometry
Background:
- Native electrospray ionization mass spectrometry (ESI-MS) is crucial for analyzing macromolecular complexes.
- Ammonium acetate, a common buffer, lacks sufficient buffering capacity at physiological pH (7.0-7.4).
- ESI-induced pH shifts can cause protein unfolding and disrupt pH-sensitive interactions.
Purpose of the Study:
- To identify and evaluate volatile, ESI-compatible buffers for maintaining physiological pH in native ESI-MS.
- To assess the buffering capacity of 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) at neutral pH.
Main Methods:
- Investigated DFEA and TFEA as potential buffers, determining their pKa values.
- Performed native ESI-MS on model proteins (cytochrome c, myoglobin) and multisubunit complexes (alcohol dehydrogenase, concanavalin A) using DFEA and TFEA.
- Conducted protein stability assays and collision-induced unfolding experiments.
Main Results:
- DFEA (pKa 7.2) and TFEA (pKa 5.5) provide buffering capacity at physiological pH, unlike ammonium acetate.
- Native ESI-MS experiments successfully preserved noncovalent protein-ligand complexes in the gas phase.
- DFEA and TFEA did not destabilize proteins in solution or the gas phase.
Conclusions:
- DFEA and TFEA are effective, ESI-compatible neutral pH buffers.
- These buffers enhance the utility of native ESI-MS for studying pH-sensitive macromolecular complexes.
- DFEA and TFEA represent a significant advancement for analyzing complex biological assemblies.
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