Mass Spectrometry Approaches for Extracting Protein Folding Energies
by Lay, Jackson O.
The model proteins Staphylococcal nuclease and ubiquitin were used to test the applicability of two H/D exchange ESI-MS methods and a protein oxidn. approach for estg. protein folding energies. These methods use the H/D exchange of globally protected amide protons or oxidn. of solvent accessible amino acids to elucidate protein folding energies. The first method, the HX-ESI-MS kinetic method is conceptually identical to SUPREX (MALDI-MS) but is based on ESI rather than MALDI. This method employs the time-dependence of H/D exchange using various denaturant concns. to ext. folding energies. The second method, the HX-ESI-MS equil. method, uses exchange at specific time points, but not the time dependence, to obtain a snapshot of open and closed populations present as a result of the protein folding equil. The equil. approach requires few assumptions in derivation of the equations used for calcn. of the folding energies. Our results support the notion that the kinetic method is useful primarily when a protein follows EX2 exchange. The equil. method, on the other hand should be applicable for either EX1 or EX2 H/D exchange mechanisms, and our data supports this contention. Finally, oxidn. can be used instead of H/D exchange to probe protein folding, and we present preliminary data showing the oxidn. of solvent accessible sites within the protein rather than H/D exchange for estg. folding energies.