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

Native mass spectrometry (nMS) is routinely used to monitor ligand binding in protein complexes. However, such interactions are easily distorted by unwanted dissociation during desolvation and ionization. This effect can be mitigated by chemical additives that modulate the ion charge, which results in lower-energy ions and better-preserved interactions. While positive-mode is the most used ionization approach, the use of negative-mode ionization has attracted attention due to its surprising ability to reduce the dissociation of some protein complexes. We therefore investigated whether charge-reducing compounds that stabilize proteins in positive ionization mode, such as imidazole, acetonitrile, and trimethylammonium-<i>N</i>-oxide, affect the charge and stability of complexes in negative ionization mode. Using the complex between myoglobin and its heme cofactor as a model system, we find that charge reduction efficiency is not maintained between positive and negative polarity, as expected for different charge reduction mechanisms. We also suggest that charge and stability correlate in positive and negative ion modes. Notably, the strongest stabilizing effects were observed for compounds that form electrospray adducts, which can dissociate during in-source activation and cool the desolvated ions. Together, the effects of charge-modulating additives suggest that relative ion stabilities may be directly comparable between polarities, although the protective effects of evaporative cooling have to be considered. We demonstrate the usefulness of the approach to detect cooperative stabilization of lysozyme by two epigallocatechin-3-gallate molecules, which requires negative ion mode and charge reduction. The strategic use of chemical additives in negative-mode ionization experiments may therefore be important for preserving complexes for analysis.