Comparison of Two ESI MS Based H/D Exchange Methods for Extracting Protein Folding Energies

Area of Science:

• Biochemistry
• Biophysics
• Analytical Chemistry

Background:

• Estimating protein folding energies is crucial for understanding protein stability and function.
• Hydrogen/deuterium exchange (HX) coupled with mass spectrometry (MS) is a powerful technique for probing protein structure and dynamics.
• Existing HX-MS methods have limitations in accuracy and applicability across different exchange mechanisms.

Purpose of the Study:

• To evaluate two novel HX electrospray ionization mass spectrometry (ESI-MS) methods for determining protein folding energies.
• To compare the performance of a kinetic-based HX ESI-MS method with an equilibrium-based method.
• To assess the reliability of these methods for proteins exhibiting different hydrogen/deuterium exchange kinetics (EX1 and EX2).

Main Methods:

• Development and application of a kinetic-based HX ESI-MS method, analogous to the Stability of Unpurified Proteins from Rates of H/D Exchange (SUPREX) method but using ESI-MS.
• Development and application of an equilibrium-based method, termed Protein Equilibrium Population Snapshot (PEPS) by HX ESI-MS, utilizing a single time point data acquisition.
• Testing both methods on model proteins: staphylococcal nuclease (known to follow EX1 kinetics) and ubiquitin (known to follow EX2 kinetics).

Main Results:

• The kinetic-based HX ESI-MS method yielded less accurate folding energy (ΔG(H2O)) and denaturant-binding (m(GdHCl)) values for staphylococcal nuclease (EX1) compared to ubiquitin (EX2).
• The PEPS method provided ΔG(H2O) and m(GdHCl) values in excellent agreement with literature data for both staphylococcal nuclease and ubiquitin.
• The study demonstrated that the PEPS method is applicable to proteins with both EX1 and EX2 hydrogen/deuterium exchange mechanisms.

Conclusions:

• The equilibrium-based PEPS method using HX ESI-MS is a robust and accurate approach for estimating protein folding energies across different exchange mechanisms.
• The kinetic-based HX ESI-MS method shows limitations when applied to proteins exhibiting EX1 exchange kinetics.
• Linear Extrapolation Method (LEM) plots showing linearity do not guarantee the validity of the obtained data.