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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Native mass spectrometry (MS) is crucial for studying protein dynamics and interactions.
• Protein sample preparation is key to maintaining native conformation and active site chemistry for MS analysis.
• Common volatile buffers like ammonium acetate may not adequately support protein conformation and ligand interactions near physiological pH.

Purpose of the Study:

• To evaluate the suitability of volatile buffer solutions for maintaining protein conformation and enzymatic activity in native MS.
• To investigate the influence of different buffer systems on protein structure, stability, and ligand interactions.
• To provide a chemical basis for optimizing buffer selection in native MS studies.

Main Methods:

• Enzymatic activity assays of cysteine desulfurase IscS in four volatile buffer solutions.
• Dual analysis of MS charge state and enzyme kinetics.
• Assessment of protein and solution physical properties.
• Comparison with traditional buffers (Tris, HEPES).

Main Results:

• Enzymatic activity of IscS in volatile buffers was comparable to traditional buffers.
• Buffer composition significantly influences protein conformation, stability, and activity.
• Specific buffers demonstrated both positive and negative effects on protein-ligand interactions and gas-phase behavior.
• A chemical rationale was established for observed buffer effects.

Conclusions:

• Native MS can identify buffer-modulated protein conformational and dynamic interactions.
• Buffer selection is critical for accurate native MS analysis of protein function and interactions.
• These findings guide the optimization of buffer systems for enhanced native MS studies.