Stabilization of Protein Interactions through Electrospray Additives in Negative Ion Mode Native Mass Spectrometry

Area of Science:

• Analytical Chemistry
• Biophysical Chemistry

Background:

• Native mass spectrometry (nMS) is vital for studying protein-ligand interactions.
• Unwanted dissociation during ionization can distort these interactions.
• Chemical additives can mitigate dissociation by modulating ion charge and energy.

Purpose of the Study:

• To investigate the effect of charge-reducing compounds, typically used in positive-mode nMS, on complex stability in negative-mode nMS.
• To compare charge reduction efficiency and stabilization mechanisms between positive and negative ionization modes.
• To explore the utility of additives in negative-mode nMS for preserving labile complexes.

Main Methods:

• Utilized myoglobin-heme cofactor complex as a model system for studying ligand binding.
• Applied various chemical additives (imidazole, acetonitrile, trimethylammonium-N-oxide) in both positive and negative ionization modes.
• Analyzed charge state and complex stability using native mass spectrometry.
• Demonstrated the approach using lysozyme and epigallocatechin-3-gallate interactions.

Main Results:

• Charge reduction efficiency differs between positive and negative ionization modes due to distinct mechanisms.
• A correlation between ion charge and stability was observed in both polarities.
• Compounds forming electrospray adducts showed strong stabilizing effects, potentially via in-source cooling.
• Relative ion stabilities are comparable between polarities when considering evaporative cooling effects.

Conclusions:

• The strategic use of chemical additives in negative-mode nMS is crucial for preserving protein complexes.
• Understanding additive effects across different polarities enhances the reliability of nMS for studying biomolecular interactions.
• This approach enables the detection of complex stabilization events, such as cooperative binding, in negative ion mode.