The Role of Nanobubbles in Protein Unfolding during Electrothermal Supercharging

Nanobubbles (NBs) are tiny gas cavities with diameters around 200 nm that remain stable in solution due to their unique properties, including low buoyancy and negative surface charges. Ammonium bicarbonate (ABC) is an alternative buffer to commonly used ammonium acetate during protein analysis by electrospray ionization (ESI) mass spectrometry. The addition of ABC under high voltage and temperature conditions can lead to protein unfolding, a phenomenon termed electrothermal supercharging (ETS). The role of CO₂ bubbles in ETS has been hypothesized and disputed. The solution stability of NBs allows for the direct observation of their effects on protein charge states and unfolding, providing insights into the potential role of CO₂ bubbles during ETS. A novel method based on flow regime switching using a Tesla valve is employed to generate stable nanobubbles in solution. NBs were also created by sonication and pressure cycling. Nitrogen and carbon dioxide nanobubbles, when produced by flow regime switching and by pressure cycling, unfold proteins such as cytochrome c and ubiquitin but not to the same extent as with ABC addition to the ESI working solution. Complete unfolding of these proteins by NBs only occurs when the ammonium ion is also present in solution. Myoglobin, known to be less structurally stable, does unfold completely under NB influence. Further, amino acids, previously shown to provide stability to proteins under ETS conditions, also prevent unfolding when NBs are present, providing additional support for the role of gas bubbles during ETS.