Solution Phase Protein Dynamics: Influences of ESI Buffers on Hydration

Abstract

Buffers are commonly selected for their compatibility with biochemical measurements or their specific capabilities, yet buffer interactions with proteins and the surrounding water can actively reshape protein activity, structure, and dynamics. Here, we compare the influences of three widely used electrospray ionization (ESI) buffers on the dynamics and stability of wild-type transthyretin (wtTTR) and TTR mutants (V30M, L55P, T119M, V122I) using native mass spectrometry (nMS). Ammonium acetate (AmAc), ethylenediammonium diacetate (EDDA), and triethylammonium acetate (TEAA) are commonly employed in nMS because they stabilize the sample in solution, facilitate gentle ionization, and minimize adduct formation on proteins and protein complexes. In comparison to AmAc, EDDA and TEAA reduce the average charge state (Zavg) of ions, consistent with conformational changes that decrease the solvent-accessible surface area (SASA) of proteins. Intact protein hydrogen–deuterium exchange experiments indicate significantly lower deuterium uptake for all TTR proteoforms in EDDA compared with AmAc or TEAA. Ion mobility MS revealed that each TTR proteoform has a larger average CCS and broader CCS distributions in AmAc, indicating greater conformational heterogeneity and dynamics than in EDDA or TEAA. Measurements of TTR tetramer disassembly and reassembly further demonstrated that the buffer identity strongly influences tetramer stability in solution. The buffer-dependent effects arise from differences in buffer-protein and buffer-solvent interactions that alter the hydration of the protein. These results highlight that buffer composition can significantly influence experimentally observed protein dynamics and stability, with important implications for interpreting measurements across biochemical and biophysical techniques.