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

## TL;DR

This study explores how different buffers affect the activity and conformation of the IscS enzyme during native mass spectrometry analyses.

## Contribution

The study reveals how volatile buffers influence enzyme activity and protein conformation in native MS.

## Key findings

- IscS enzymatic activity is comparable in volatile buffers to traditional buffers like Tris and HEPES.
- Buffer systems significantly modulate protein conformation and stability in native MS.
- MS charge state and enzyme kinetics are influenced by buffer properties.

## Abstract

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.