Probing Protein Denaturation during Size-Exclusion Chromatography Using Native Mass Spectrometry

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Protein Analysis

Background:

• Size-exclusion chromatography coupled with electrospray-ionization mass spectrometry (SEC-ESI-MS) analyzes proteins in their native state.
• Assessing eluent effects on protein-stationary phase interactions and denaturation is often overlooked.

Purpose of the Study:

• Investigate the impact of volatile mobile phase additives on SEC retention and ESI of proteins.
• Determine optimal conditions to maintain protein integrity during SEC-ESI-MS analysis.

Main Methods:

• Utilized myoglobin as a model protein, varying eluent ionic strength and pH.
• Calculated SEC distribution coefficient to assess protein-stationary phase interactions.
• Analyzed protein-ion charge state distributions using native ESI-MS to monitor structural changes.

Main Results:

• Most tested eluents caused nonideal SEC behavior or protein unfolding.
• Ammonium acetate above 0.1 M ionic strength prevented protein-stationary phase interactions.
• Ammonium acetate (kosmotropic) prevented denaturation, unlike ammonium formate and bicarbonate (chaotropic).
• 0.2 M ammonium acetate (pH 6.9) enabled native SEC-ESI-MS analysis of multiple proteins across different columns.

Conclusions:

• Mobile phase composition significantly impacts protein retention and structure in SEC-ESI-MS.
• Ammonium acetate is a suitable mobile phase additive for preserving protein native states.
• SEC-ESI-MS can reveal subtle protein structural changes and interactions.