Date of Graduation


Document Type


Degree Name

Bachelor of Science

Degree Level



Chemistry & Biochemistry


Stites, Wesley

Committee Member/Reader

Liyanage, Rohana

Committee Member/Second Reader

Henry, Ralph

Committee Member/Third Reader

Goodman-strauss, Chaim


In this study, Protein Equilibrium Population Snapshot Hydrogen-Deuterium Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) was applied to study the local regions of model proteins, staphylococcal nuclease and ubiquitin. The hydrogen deuterium exchange (HDX) has become a key technique for studying the structural and dynamic aspects of proteins in solution. This technique creates a rapid exchange between all of the exchangeable hydrogen ions with deuterium when the protein is exposed to a solvent. The PEPS method is an equilibrium-based method used to determine the populations of the closed native and open denatured states of a protein. By combining the applications of HDX and the PEPS method using ESI-MS, one can determine the solvent accessible and protected amide protons, the folding energies and rates of a protein in physiological conditions through linearly extrapolating the folding energies, and the rates by systematically denaturing conditions using high guanidine hydrochloride (GdHCl).

Past studies have applied this method on intact model proteins, staphylococcal nuclease and ubiquitin. Both proteins showed the expected amount of exchangeable amide protons, which was verified from the X-ray structures. The folding energies, folding rates, and unfolding rates for staphylococcal nuclease and ubiquitin were estimated to be -4.8 kcal mol-1, 10 s-1, 2x10-3 s-1 and -8.8 kcal/mol, 251 s-1, 4x10-5 s-1 respectively. This work successfully deconvoluted local HDX coverage, local folding energies, and local folding rates from this intact protein information. This was accomplished by dissecting or digesting the intact protein using pepsin after the HDX without losing already incorporated deuterium under HDX quenching conditions, at pH 2.7 and 0o C followed by LC-ESI-MS analysis. These results were compared to the results of the local regions to investigate how independent they were to the intact proteins.

The same procedure applied in the past study involving the intact proteins was applied in this study but with a pepsin digestion. This digestion was performed after the HDX, but before the LC-ESI-MS analysis. The resulted pepsin fragments contained all of the necessary information for the folding energies, folding rates, and accessible areas for local regions. Control experiments were performed to identify the local regions using multiple mass spectrometry methods. The intact peptide mass was searched on an available software of in silico pepsin digestion of the protein being analyzed. For further verification, fragmentation was applied with LC-MS/MS, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), and matrix-assisted laser desorption/ionization Fourier transfer mass spectrometry (MALDI-FT-MS).

Local regions of the staphylococcal nuclease had energies between -3 to -5 kcal/mol, and the folding rates ranged from 0.01 to 10 s-1. Unfolding rates on average seemed to be in close proximity, 1.5x10-4 s-1. This was probably consistent with the cooperativity in unfolding proteins. The local regions of ubiquitin had energies ranging from -1 to -10 kcal/mol, folding rates ranged from 10-3 -108 s-1. Again, similar to staphylococcal nuclease, ubiquitin also has on average similar unfolding rates, ~ 10-5 s-1.

This study attempted to answer several important questions. For example, how much influence and independence do the local secondary structures (alpha helices, beta sheets, and loops) have on the intact protein properties? Can the intact protein structure be constructed in cases where the folding and unfolding properties of local regions are not known? Can a database be constructed in which it has experimental sequence specific folding and unfolding properties?