Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

Native mass spectrometry paired with ion mobility (IM-MS) provides the capacity to monitor the structure of protein complexes and simultaneously assess small molecule binding to the protein. Native IM-MS typically utilizes positive mode electrospray ionization producing a distribution of multiply charged protein species. For membrane proteins, these charge states are often too high resulting in protein gas phase unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, the utility of alkali metal salts as a charge reducing additive was explored. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, ELIC. The charge reducing effect was only present in membrane proteins, and could not be accounted for by conformational changes in ELIC structure. Charge reduction by alkali metal salts was also detergent dependent, and was most pronounced in long PEG-based detergents such as C10E5 and C12E8. Based on these results, a mechanism was posited for alkali metal charge reduction of membrane proteins. Addition of low concentration of alkali metals may provide an advantageous approach for charge reduction of detergent solubilized membrane proteins by native MS.