IscS Kinetics in Native Mass Spectrometry Buffers Reveal Key Physiochemical Properties that Influence Enzyme Activity.

Investigations of protein function and interactions with native mass spectrometry (MS) have yielded significant insights into protein dynamics, transient reaction intermediates, and pharmacokinetic targets. The pursuit of these studies and their outcomes depend on the preparation of protein samples in a manner able to support native conformation, active site chemistry, and protein-ligand interactions. Although ammonium acetate is a commonly utilized volatile buffer in MS-based analyses, the gap in buffer capacity near physiological pH calls into question whether this or other volatile buffer solutions are able to facilitate native conformation and protein-ligand interactions in the gas phase. We report enzymatic activity of the cysteine desulfurase IscS in four volatile buffer solutions comparable to that observed in traditionally utilized buffers such as Tris and HEPES, which is heavily influenced by buffer contributions to protein conformation and stability. We present a dual analysis of MS charge state and enzyme kinetics in the context of protein and solution physical properties, providing a chemical justification for the positive and negative effects of specific buffers. Ultimately, these results demonstrate how native MS technology can be used to identify protein conformational and dynamic interactions modulated by buffer systems to guide mechanistic studies.