<p>Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.</p> / Dissertation

<p>While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment. </p><p>The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment. </p><p>The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.</p> / Dissertation