Browsing by Author "Fitzgerald, Michael C"

Currently there is a dearth of analytical techniques for studying protein-ligand interactions on the proteomic scale. Existing techniques, which rely on various calorimetry or spectroscopy methods, are limited in their application to the proteomic scale due to their need for large amounts of pure protein. Recently, several mass spectrometry-based methods have been developed to study protein-ligand interactions. These mass spectrometry-based methods overcome some of the limitations of existing techniques by enabling the analysis of unpurified protein samples. However, the existing mass spectrometry-based methodologies for the analysis of protein-ligand binding interactions are not directly compatible with current mass spectrometry-based proteomics platforms.

Described here is the development and application of a new technique designed to detect and quantify protein-ligand binding interactions with mass spectrometry-based proteomic platforms. This technique, termed SPROX (Stability of Proteins from Rates of Oxidation), uses an irreversible covalent oxidation labeling reaction to monitor the global unfolding reactions of proteins to measure protein thermodynamic stability. Two variations of the SPROX technique are established here, including one variation that utilizes chemical denaturant to induce protein unfolding and a second variation that utilizes temperature to denature proteins. The SPROX methodology is tested on five proteins including ubiquitin, ribonuclease A, bovine carbonic anhydrase II, cyclophilin A, and calmodulin. Results obtained on these model systems are used to determine the method's ability to measure the thermodynamic parameters associated with each protein's folding/unfolding reaction. Results obtained on calmodulin and cyclophilin A are used to determine the method's ability to quantify the dissociation constants of protein-ligand complexes.

The primary motivation for the development of the SPROX protocols in this work was to create a protein-ligand binding assay that could be interfaced with conventional mass spectrometry-based platforms. Two specific SPROX protocols, including a label-free approach and an oxygen-16/18 labeling approach, are developed and demonstrated using the thermal SPROX technique to analyze ligand binding in a model four-protein component mixture consisting of ubiquitin, ribonuclease A, bovine carbonic anhydrase, and cyclophilin A. The thermal SPROX technique's ability to detect cyclosporin A binding to cyclophilin A in the context of the model mixture is shown using both labeling approaches.

An application using the SPROX technique combined with a multi-dimensional protein identification technology (MudPIT)-based proteomics platform is also described. In this application, which utilized an isobaric mass tagging strategy, 325 proteins in a yeast cell lysate are simultaneously assayed for CsA-binding. This study was also used to investigate the protein targets of an already well-studied immunosuppressive drug, cyclosporin A. Two of the ten protein targets identified in this work are known to interact with CsA, one through a direct binding event and one through an indirect binding event. The eight newly discovered protein targets of CsA suggest a molecular basis for post-transplant diabetes mellitus, which is a side effect of CsA in humans.

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.

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.

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.

Recently, several mass spectrometry-based proteomics techniques have been developed for the large-scale analysis of thermodynamic measurements of protein stability. This has created the possibility of characterizing disease states via differential thermodynamic stability profiles. Described here is the application of the Stability of Proteins from Rates of Oxidation (SPROX) technique to characterize mouse models of disease. The mouse models studied here are of normal aging and two genetically induced Parkinson’s Disease (PD) models.

Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from C57BL/6 mice at age 6- (n=7) and 18-months (n=9). The biological variability of the protein stability measurements was low, and within the experimental error of the SPROX technique. Remarkably, the large majority of the 83 brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p<0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related disease.

One of the PD mouse models involved characterizing the protein interactions induced by mutated leuceine-rich repeat kinase 2 (LRRK2) at a pre-symptomatic time point (3 months old). The models used were a control, overexpressed wildtype LRRK2, and overexpressed R1441G mutated LRRK2 (n=2 for all models). Comparative analyses on thermodynamic stability profiles of ~470 proteins revealed relatively few differences. In fact, the observed hit rate in each comparative analysis was close to that associated with the biological variability of the mice. However, four protein hits, dihydropyrimidinase-related protein 2, eukaryotic translation initiation factor 4A2, Rap1 GTP-GDP dissociation stimulator 1 and myelin basic protein, were identified with consistent thermodynamic stability in multiple mice within a biological state and as hits in multiple comparisons suggesting they are the most likely to be true positives.

The second PD mouse model studied was one in which the human α-synuclein protein, containing the known PD mutation A53T, was overexpressed. To characterize the disease progression of PD induced by this mutation, mice were sacrificed at 1 month (n=4), 6 months (n=4) and when they became symptomatic at 10-16 months (n=3). Thermodynamic stability profiles were generated for >850 proteins at each time point. The relative stabilities of these proteins were assayed in a series of comparative analyses involving mice at the different time points and the normally aged mice from above. In total 244 peptides were found to be differentially stabilized during PD progression. A subset of 52 peptide hits was identified to be of particular interest. Of these 52 peptides 22 were identified with early disease progression, 5 peptides showed late disease progression, 5 peptides reported a gradual difference in stability over disease progression and 20 peptides indicated no disease progression trend. More than 90% of the 32 peptides indicating a trend in disease progression showed progression related destabilization.

The results of this thesis help validate the use of thermodynamic stability measurements to capture disease-related proteomic differences in mice. Furthermore, these results establish a new biophysical link between the hit proteins identified and their role in aging, LRRK2 protein interactions, and PD progression.

In the past decade, several mass spectrometry-based proteomic techniques have been developed for the large-scale analysis of protein folding stabilities. The main focus of this dissertation is to develop and apply these large-scale protein folding stability approaches in drug target identification and disease biomarker discovery. One goal of this work is to develop a novel chemo-selection strategy to improve the bottom-up proteomics readout in proteome-wide limited proteolysis experiments. Another goal of this work is to apply these methods to the target identification of two drugs with known mode of action, and to the biomarker discovery of Parkinson’s disease.

Described in the first part of the dissertation is the development of a chemo-selective enrichment strategy to isolate the semi-tryptic peptides generated in mass spectrometry-based applications of limited proteolysis methods. The method is termed Semi-Tryptic Peptide Enrichment Strategy for Proteolysis Procedures (STEPP). The STEPP-PP workflow was evaluated in two proof-of-principle drug target identification experiments involving two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow was evaluated in one proof-of-principle experiment on identification of protein conformational changes between a breast cancer cell line, MCF-7, and a normal cell line, MCF-10A. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities. When coupled to a one-pot data acquisition strategy, the one-pot STEPP-PP technique was found to have a very low false positive rate (i.e., 0.09%) in a proof-of-principle drug target identification experiments involving cyclosporin A and a yeast lysate.

The second part of this dissertation describes the application of protein folding stability approaches to the identification of protein targets of subglutinol A (a natural immunosuppressant) and manassantin A (a natural product with anti-cancer activity).

In the suglutinol A study, a combination of SPROX, TPP, CPP and STEPP-PP strategies was used to identified two consistent protein hits, deoxycytidine kinase (DCK) and exportin-2 (XPO2), from more than 2000 assayed proteins in a 2B4T cell lysate. The binding of DCK with subglutinol A was validated using a targeted gel-based pulse proteolysis experiment. A set of chemical biology experiments were performed to uncover the relation of this interaction with subglutinol A’s mode of action. It was shown that neither of the kinase activity, expression level or phosphorylation modification level of DCK was alternated by subglutinol A. However, the nuclear transportation of DCK was blocked by subgltutinol A. This reduction of DCK level in the cell nucleus possibly leads to the observed reduction of nuclear dCMP pool and the halted proliferation of sublgutinol A treated T cells.

In the manassantin A study, a combination of STEPP-LiP, STEPP-PP, one-pot STEPP-PP, one-pot SPROX and one-pot TPP strategies were performed to identify the protein target of the drug in a hypoxia-treated HEK293T cell lysate. These experiments assayed over 4000 proteins and found 4 protein hits for further validation of their interaction with manassantin A.

The third part of describes the utilization of the SPROX method to characterize the progression of PD in a mouse model of the disease in which the human α-synuclein protein with an A53T mutation was overexpressed. The thermodynamic stabilities of proteins in brain tissue cell lysates from Huα-Syn(A53T) transgenic mice were profiled at three time points including at 1 Month (n=9), at 6 Months (n=7), and at the time (between 9 and 16 Months) a mouse became Symptomatic (n=8). The thermodynamic stability profiles generated here on over 300 proteins were compared to the thermodynamic stability profiles generated on the same proteins from similarly aged wild-type mice using a two-way ANOVA analysis. A group of 22 proteins were identified with age-related protein stability changes, and a group of 11 proteins were found to be differential stabilized in the Huα-Syn(A53T) transgenic mouse model. The proteins differentially stabilized in the disease mouse model could potentially be used as Parkinson’s disease biomarkers upon further validation.

Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.

This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.

The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.

To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.

The identification and quantification of protein-protein interactions in large scale is critical to understanding biological processes at a systems level. Current approaches for the analysis of protein -protein interactions are generally not quantitative and largely limited to certain types of interactions such as binary and strong binding interactions. They also have high false-positive and false-negative rates. Described here is the development of and application of mass spectrometry-based proteomics metehods to detect and quantify the strength of protein-protein and protein-ligand interactions in the context of their interaction networks. Characterization of protein-protein and protein-ligand interactions can directly benefit diseased state analyses and drug discovery efforts.

The methodologies and protocols developed and applied in this work are all related to the Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX) and Stability of Protein from Rates of Oxidation (SPROX) techniques, which have been previously established for the thermodynamic analysis of protein folding reactions and protein-ligand binding interactions. The work in this thesis is comprised of four parts. Part I involves the development of a Histidine Slow H/D exchange protocol to facility SURPEX-like measurements on the proteomic scale. The Histidine Slow H/D exchange protocol is developed in the context of selected model protein systems and used to investigate the thermodynamic properties of proteins in a yeast cell lysate.

In Part II an isobaric mass tagging strategy is used in combination with SPROX (i.e., a so-called iTRAQ-SPROX protocol) is used to characterize the altered protein interactions networks associated with lung cancer. This work involved differential thermodynamic analyses on the proteins in two different cell lines, including ADLC-5M2 and ADLC-5M2-C2.

Parts III and IV of this thesis describe the development and application of a SPROX protocol for proteome-wide thermodynamic analyses that involves the use of Stable Isotope Labeling by Amino acid in cell Culture (SILAC) quantitation. A solution-based SILAC-SPROX protocol is described in Part III and a SILAC-SPROX protocol involving the use of cyanogen bromide and a gel-based fractionation step is described in Part IV. The SILAC-SPROX-Cyanogen bromide (SILAC-SPROX-CnBr) protocol is demonstrated to significantly improve the peptide and protein coverage in proteome-wide SPROX experiments. Both the SILAC-SPROX and SILAC-SPROX-CnBr porotocols were used to characterize the ATP binding properties of yeast proteins. Ultimately, the two protocols enabled 526 yeast proteins to be assayed for binding to AMP-PNP, an ATP mimic. A total of 140 proteins, including 37 known ATP-binding proteins, were found to have ATP binding interactions.

SPROX (Stability of Proteins from Rates of Oxidation) is a technique for the detection and quantitation of protein-ligand binding interactions. In SPROX the differential denaturant dependence of a methionine oxidation reaction in proteins performed in the presence and in the absence of ligand is used to measure the thermodynamic properties of protein-ligand binding interactions. Recently, the SPROX technique has also been used with quantitative mass spectrometry-based proteomic strategies to simultaneously assay up to hundreds of different proteins in complex biological samples. The proteomic applications of SPROX, to date, have relied on the use of a methionine-containing-peptide enhancement strategy and 1-D liquid chromatography-tandem mass spectrometry (LC-MS/MS) and. The strategies presented here, including gel electrophoresis fractionation and gas-phase fractionation of derivatized methionine-containing peptides, aim to increase proteomic coverage in SPROX by facilitating the detection, identification, and quantitation of methionine-containing peptides in the proteomics experiment. The initial results obtained in this work have shown that gel electrophoresis can help increase the proteome coverage while identifying complementary peptides to the current solution-based SPROX analysis. A gas-phase fractionation strategy is also demonstrated; however, the mass spectrometric analysis needs to be optimized and further tested to achieve selectivity for methionine peptides. Both of these fractionation strategies show promise for improving the proteomic coverage in SPROX studies of protein-ligand interactions on the proteomic scale. In addition, the previously established SPROX protocol using isobaric mass tags was used in two protein-ligand binding experiments, including one experiment to identify the protein targets in a yeast cell lysate of adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP), a non-hydrolyzable adenosine-5'-triphosphate (ATP) mimic, and second experiment to investigate the protein targets of two iron chelators, deferasirox (Exjade) and N'-[1-(2hydroxyphenyl) ethyliden]isonicotinoylhydrazide (HAPI), both of which protect ARPE-19 cells from oxidative damage induced by hydrogen peroxide. Three known ATP-binding proteins, ADEnine requiring, URAcil requiring and Yeast Elongation Factor, have been identified, while further investigation for the protein targets of iron chelators needs to be performed.

Protein stability profiling techniques, including stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and limited proteolysis (LiP), have created a new toolbox for making protein folding stability measurements on the proteomic scale. Despite the broad usage of these techniques in an increasingly large number of applications, few studies have been reported in which more than one of these techniques have been used for the same application. Therefore, detailed information about the complementarity of these different experimental approaches is lacking. A major focus of the work described in this thesis is the comparative analysis of these different experimental strategies in different application areas. Described in Chapter 2 of this dissertation is a new experimental workflow for multiplexing TPP analyses that is also amenable to SPROX analyses. In a proof-of-principle study, this new workflow (termed, "one-pot" workflow) was demonstrated to significantly reduce the instrument time and cost of materials by 10-fold, which consequentially enabled enhanced multiplexing compared to traditional protein stability profiling analysis. This "one-pot" workflow, which is also amenable to SPROX analysis, makes feasible the acquisition of protein stability profiling data from multiple biological replicates and even multiple techniques (e.g., SPROX and TPP) in a given application.

Described in Chapters 3, 4, and 5 of this dissertation are applications of SPROX, TPP, and LiP to a series of protein target discovery projects involving several different small molecule drugs with known biological activities but unknown modes of action. These applications included the use of multiple energetics-based techniques and in some cases included the "one-pot" workflow, to identify the protein targets of the bioactive small molecules. These results of these studies included: i) identification of TCP-1 as the anti-malarial target of clemastine in a Plasmodium falciparum (Pf) cell lysate; ii) identification of potential Pf protein targets of several additional anti-malarial drugs; iii) identification of Rab-1A as the anti-Parkinson target of NAB1; iv) identification of potential protein targets of anti-salmonella drugs in a complex host-pathogen system; and v) identification of the interactome of CoA and de-phosphorylated CoA in mammalian cells. The results of these protein target discovery studies not only helped elucidate the molecular basis of the biological activities of the above small molecules, but the results also demonstrated the power of using multiple protein stability profiling techniques to deconvolute protein targets and identify small molecule interactomes.

The final chapter of this dissertation describes the use of protein stability profiling methods, including the "one-pot" SPROX and TPP workflows as well as LiP, to identify potential prognostic biomarkers of oxaliplatin resistance in colorectal cancer. The goal of this work was to identify the overlapping protein hits identified in SPROX, TPP, and LiP analyses of oxaliplatin resistant and sensitive colorectal cancer cell lines (n=3 cell line comparisons) and patient-derived xenograft mouse models of oxaliplatin resistance and sensitivity (n= 1 mouse model comparison). Ultimately, 23 differentially stabilized proteins were identified with differential stability in at least 2 techniques and consistent results in both cell culture models and PDX mouse models. These 23 proteins included 9 proteins being previously connected to cancer chemo-resistance. One of these 9 differentially stabilized proteins, major vault protein (MVP), was further investigated to elucidate its role on colorectal cancer chemoresistance. This research demonstrated that complementary protein stability profiling is a complementary approach to identify potential prognostic biomarkers and shed light on understanding the molecular mechanism of colorectal cancer chemoresistance.

In conclusion, the work in this dissertation described an integrated workflow for energetics-based proteomic techniques that can be used for target deconvolution and biomarker identification in complex biological systems.

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.

The characterization of protein stability changes and protein-ligand interactions on the proteomic scale is important for understanding the biology of cellular processes. The identification and quantification of protein-ligand binding affinities is critical for disease state analyses and drug discovery. A mass spectrometry-based technique, Stability of Proteins from Rates of Oxidation (SPROX), has been established for the thermodynamic analysis of protein stability and protein-ligand interactions. In the first part of this dissertation, a previously published iTRAQ-SPROX protocol is improved by incorporating a filter assisted sample preparation (FASP) protocol to significantly reduce sample loss during the experiment. Also, in order to eliminate methionine as a potential contaminant that can cause signal suppression during LC-MS/MS analysis, TCEP•HCl is used to quench the H2O2 oxidation instead of methionine. This avoids the potential reaction between the free methionine and the iTRAQ reagents. The improved protocol, referred to hereafter as the iTRAQ-FASP-SPROX protocol, is shown to increase the peptide/protein coverages for less concentrated cell lysate samples, and it is applied here to study the protein-ligand interaction networks between human ARPE-19 cells lysis and two different iron chelators (HAPI and Exjade). Information on potential protein targets of these two iron chelators are reported.

In the second part of this dissertation, a targeted MS-based approach for protein-ligand binding analysis is developed to analyze targeted subsets of proteins in a proteome. The so-called PAB-SPROX protocol is demonstrated to be applicable for the detection and relative quantitation of targeted methionine-containing peptides in +/- ligand samples by using isotopically labeled light and heavy PAB (i.e. 12C6-PAB and 13C6-PAB). Multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods are demonstrated to be amenable to PAB-SPROX analyses. In addition to proof-of-principle studies involving the cylclophilin A-cyclosporine A binding interaction, the PAB-SPROX protocol was used to validate the direct interaction between YBX1 protein and tamoxifen using very limited amount of purified YBX1 protein. Applications of PAB-SPROX protocol have also included the validation of potential binding targets of Staurosporine, Manassatin A and Tamoxifen. The PAB-SPROX studies with these latter ligands facilitated the identification of false positives in previous proteome-wide SRPOX studies.

The direct link between a protein’s thermodynamic stability and function influenced the development of mass spectrometry-based methods to characterize the energetics associated with protein folding that enabled the large-scale elucidation of drug protein targets and disease state protein biomarkers. This area of structural biology is undergoing constant development and new applications are emerging. Consequently, the original contributions of this dissertation include (1) the continuation or extension of mass spectrometry and energetic-based strategies for proteome-wide characterization of protein folding stabilities in allergen-containing proteomes to discriminate allergenicity, (2) the hybridization of novel strategies with existing energetic-based approaches utilizing mass spectrometry readout for simpler and efficient characterization of protein folding stabilities and ligand binding, and (3) the development of novel mass spectrometry-based strategies for comprehensive evaluations of the thermodynamics and kinetics of protein folding.First, this dissertation describes comprehensive protein profiling methods to discriminate allergens from non-allergens. As continuation, RNA sequencing (RNA-seq) analysis served as a proxy for protein abundance, and the Stability of Proteins from Rates of Oxidation (SPROX) reported on thermodynamic stability. These techniques characterized the protein expression levels and stability of proteins in the European white birch pollen, Betula pendula (Bp), and German cockroach, Blattella germanica (Bg). The simultaneous comparison of stability and abundance confirmed that Bp and Bg allergens had significantly higher expression levels and higher stabilities compared to non-allergens from the same source. Combining the Bp and Bg results with previous studies for a robust statistical comparison of the abundance and stability of allergens and non-allergens from indoor and outdoor sources confirmed that allergens were significantly more abundant and more stable. The thermodynamic stability of the proteins in Bp was further investigated utilizing a denaturant-dependent Pulse Proteolysis (PP) strategy with thermolysin. Additionally, proteolytic susceptibility was assessed by employing a time-dependent cathepsin S digestion under native conditions. The results confirmed that allergens were significantly less susceptible to thermolysin (more thermodynamically stable) or cathepsin S digestion than the non-allergens in Bp. Additionally, no correlation resulted between the SPROX- and PP-derived thermodynamic stabilities and between the thermodynamic stabilities and proteolytic susceptibilities of selected proteins from Bp. The absence of correlation is attributed to the fundamental differences between techniques—each technique utilizes distinct probes to report on a protein’s thermodynamic stability and/or proteolytic susceptibility. Finally, the PP-derived stability for the major Bp allergen, Bet v 1, correlated with the LiP-derived proteolytic susceptibility and the generation of known T-cell epitopes connecting stability with endosomal processing having allergenic or immunogenic implications. Next, this dissertation reports the first application of the novel one-pot analysis in conjunction with the SPROX methodology for a simplified and efficient evaluation of protein folding and ligand-binding. A hybrid of the one-pot analysis with SPROX utilizing a MALDI readout enabled efficient evaluations of protein stability and ligand binding. The approach generated protein folding stabilities with similar precision to the standard curve-fitting SPROX technique. Furthermore, the one-pot analysis was coupled with the SPROX strategy for a comprehensive deconvolution of Cyclosporine A (CsA) protein targets in yeast. This novel approach identified 3 known CsA protein hits with a 0.04% false positive rate. A cross-validation between techniques (i.e., TPP, CPP, or PP, performed under similar conditions) resulted in false positive rates approaching 0 %. Finally, this dissertation showcases the development of a novel approach utilizing a native or low denaturant-based Reagent-dependent Thiolate-based Reactivity (RTR) assay utilizing mass spectrometry for the evaluation of the thermodynamics and kinetics of protein folding. An RTR strategy titled MTR utilizing a MALDI readout was performed under native conditions to report on the thermodynamics of protein folding. The MTR strategy measured the thermodynamic stability of mutants of the C domain of protein A from Staphylococcus aureus. Additionally, a low denaturant MTR approach reported the thermodynamics and kinetics of protein folding for bovine β-lactoglobulin B (LG-B). A comprehensive application of the native RTR approach was performed on yeast providing thermodynamic stability information for a subset of the proteins.

Proteins fold into well-defined three-dimensional structures to carry out their biological functions which involve non-covalent interactions with other cellular molecules. Knowledge about the thermodynamic properties of proteins and protein-ligand complexes is essential for answering fundamental biological questions and drug or biomarker discovery. Recently, chemical labeling strategies have been combined with mass spectrometry methods to generate thermodynamic information about protein folding and ligand binding interactions. The work in this thesis is focused on the development and application of two such chemical labeling protocols coupled with mass spectrometry including one termed, SUPREX (stability of unpurified proteins from rates of H/D exchange), and one termed SPROX (stability of proteins from rates of oxidation). The work described in this thesis is divided into two parts. The first part involves the application of SUPREX to the thermodynamic analysis of a protein folding chaperone, Hsp33, and its interaction with unfolded protein substrates. The second part involves the development of a new chemical labeling protocol that can be used to make protein folding and ligand binding measurements on the proteomic scale.

In the first part of this work, the SUPREX technique was used to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of the studies, which were performed at the intact protein level, suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived Kd-values for Hsp33 complexes with four different substrates were also found to be all within a range of 3-300 nM. The interaction between Hsp33 and one of its substrates, citrate synthase (CS), was characterized at a higher structural resolution by using the SUPREX technique in combination with a protease digestion protocol. Using this protocol, the thermodynamic properties for both Hsp33 and CS were evaluated at different stages of binding, including reduced Hsp33 (inactive form), oxidized Hsp33 (active form), followed by native CS and finally of Hsp33ox -CS complexes before and after reduction with DTT. The results suggest that Hsp33 binds unfolded proteins that still have a significant amount of residual higher- order structure. Structural rearrangements of the substrate protein appear to occur upon reduction of the Hsp33-substrate complexes, which may facilitate the transfer of the substrate protein to other protein folding chaperone systems.

In the second part of this dissertation, a mass spectrometry-based covalent labeling protocol, which relies on the amidination rate of globally protected protein amine groups, was designed and applied to the thermodynamic analysis of several eight protein samples including: six purified proteins (ubiquitin, BCAII, RNaseA, 4OT, and lysozyme with, and without GlcNAc), a five-protein mixture comprised of ubiquitin, BCAII, RNaseA, Cytochome C, and lysozyme, and a yeast cell lysate. The results demonstrate that in ideal cases the folding free energies of proteins and the dissociation constants of protein-ligand complexes can be accurately evaluated using the protocol. Also demonstrated is the new method's compatibility with three different mass spectrometry-based readouts including an intact protein readout using MALDI, a gel-based proteomics readout using MALDI, and an LC-MS-based proteomics readout using isobaric mass tags. The results of the cell lysate sample analysis highlight the complementarity of the labeling protocol to other chemical modification strategies that have been recently developed to make thermodynamic measurements of protein folding and stability on the proteomic scale.

Protein-ligand interactions can be detected and quantified using protein folding stability measurements. Thus, protein folding stability changes are closely linked to protein function and are an important biophysical measurement. Stability of Proteins from Rates of Oxidation (SPROX) is one approach for making proteome wide stability measurements on proteins. Previous SPROX data analysis strategies relied heavily on visual inspection of the data to identify protein stability changes. This created a bottleneck in the analysis and chances for human error. As part of this work, several new data analysis strategies were evaluated using data from previously reported ligand-binding studies. One strategy, the so-called difference analysis, was determined to be the data analysis strategy of choice as it minimized false positive and negatives. The difference analysis strategy was applied to identify protein targets of the breast cancer therapeutic tamoxifen (TAM) and its active metabolite 4-hydroxytamoxifen (4OHT) in yeast and protein targets of TAM and its most abundant metabolite n-desmethyl tamoxifen (NDT) in MCF-7 cell lysate. Several yeast protein targets of TAM and 4OHT were identified using SPROX, and they were subsequently validated using a pulse proteolysis strategy. The proteins in an MCF-7 cell line were probed for TAM- and NDT-induced stability changes using the SPROX in combination with two different quantitative proteomics strategies. Together the SPROX experiments on the proteins in an MCF-7 cell lysate enabled over 1000 proteins to be assayed for TAM- and NDT- induced protein stability changes. Ultimately, a total of 163 and 200 proteins with TAM- and NDT-induced stability changes were identified, respectively. A subset of 33 high confidence hits, including those identified using both proteomics strategies or those identified with multiples peptide probes, were assessed for experimental links to the ER using a STRING analysis. One high confidence protein hit, Y-box binding protein 1 (YBX1), was recently shown to bind the estrogen receptor, which is the known target of TAM. Preliminary results generated here using pulse proteolysis and a purified recombinant YBX1 protein construct suggest that YBX1 is a direct protein target of TAM. Proteins with altered expression levels with TAM and NDT treatment were also identified here. In total, 799 and 671 proteins were probed for TAM- and NDT- induced expression changes, respectively, and 49 and 30 proteins had altered expression. Out of the 49 and 30 proteins with TAM- and NDT- induced expression changes, 14 and 4 proteins had TAM- and NDT- induced altered stability, respectively. In addition to the above ligand-binding studies, SPROX was utilized to characterize the stability of the allergen containing proteomes of the European house dust mite, timothy grass pollen, and ragweed pollen. It was determined that the protein allergens in these proteomes were more stable and more abundant (based on transcriptomic data), than non-allergen proteins from these sources.

Detection and quantification of protein-ligand binding interactions is extremely important for understanding interactions that occur in biological systems. Since traditional techniques for characterizing these types of interactions cannot be performed in complex systems such as cell lysates, a series of energetics-based techniques that are capable of assessing protein stability and measuring ligand binding affinities have been developed to overcome some of the limitations of previous techniques. Now that the capabilities of the energetics-based techniques have been exhibited in model systems, the false-positive rates of the techniques, the range of biological questions to which the techniques can be addressed, and the use of the techniques to discover novel interactions in unknown systems remained to be shown. The Stability of Proteins from Rates of Oxidation (SPROX) technique and the Pulse Proteolysis (PP) technique were applied to a wide range of biological questions in both yeast and human cell lysates to evaluate the scope of these experimental workflows. The false-positive rate of iTRAQ-SPROX protein target discovery on orbitrap mass spectrometer systems was determined to be < 0.8 %. The iTRAQ-SPROX technique was successfully applied to the discovery of both known and novel protein-protein, protein-ATP, and protein-drug interactions, leading to the quantification of protein-ligand binding affinities in each of these studies. In the pursuit of discovering geldanamycin protein interactors, the use of iTRAQ-SPROX and SILAC-PP in combination was determined to be advantageous for confirming protein-ligand interactions since the techniques utilize different quantitation strategies that are subject to separate technical errors in quantitation. Finally, the iTRAQ-SPROX and SILAC-PP techniques were used to evaluate the interactions of manassantin A in a human cell lysate. In this work, a previously unknown protein target of manassantin A, Filamin A, was detected as a hit protein using both the iTRAQ-SPROX and SILAC-PP protocols. The work completed in this dissertation has expanded the understanding of the limitations of energetics-based techniques and shown that biological replicate analyses are essential to confirm ligand interactions with novel protein targets.

Protein biomarkers can facilitate the diagnosis of many diseases such as cancer and they can be important for the development of effective therapeutic interventions. Current large-scale biomarker discovery and disease state characterization studies have largely focused on the global analysis of gene and protein expression levels, which are not directly tied to function. Moreover, functionally significant proteins with similar expression levels go undetected in the current paradigm of using gene and protein expression level analyses for protein biomarker discovery. Protein-ligand interactions play an important role in biological processes. A number of diseases such as cancer are reported to have altered protein interaction networks. Current understanding of biophysical properties and consequences of altered protein interaction network in disease state is limited due to the lack of reproducible and high-throughput methods to make such measurements. Thermodynamic stability measurements can report on a wide range of biologically significant phenomena (e.g., point mutations, post-translational modifications, and new or altered binding interactions with cellular ligands) associated with proteins in different disease states. Investigated here is the use of thermodynamic stability measurements to probe the altered interaction networks and functions of proteins in disease states. This thesis outlines the development and application of mass spectrometry based methods for making proteome-wide thermodynamic measurements of protein stability in multifactorial complex diseases such as cancer. Initial work involved the development of SILAC-SPROX and SILAC-PP approaches for thermodynamic stability measurements in proof-of-concept studies with two test ligands, CsA and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). In these proof-of-principle studies, known direct binding target of CsA, cyclophilin A, was successfully identified and quantified. Similarly a number of known and previously unknown ATP binding proteins were also detected and quantified using these SILAC-based energetics approaches.

Subsequent studies in this thesis involved thermodynamic stability measurements of proteins in the breast cancer cell line models to differentiate disease states. Using the SILAC-SPROX, ~800 proteins were assayed for changes in their protein folding behavior in three different cell line models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. Approximately, 10-12% of the assayed proteins in the comparative analyses performed here exhibited differential stability in cell lysates prepared from the different cell lines. Thermodynamic profiling differences of 28 proteins identified with SILAC-SPROX strategy in MCF-10A versus MCF-7 cell line comparison were also confirmed with SILAC-PP technique. The thermodynamic analyses performed here enabled the non-tumorigenic MCF-10A breast cell line to be differentiated from the MCF-7 and MDA-MB-231 breast cancer cell lines. Differentiation of the less invasive MCF-7 breast cancer cell line from the more highly invasive MDA-MB-231 breast cancer cell line was also possible using thermodynamic stability measurements. The differentially stabilized protein hits in these studies encompassed those with a wide range of functions and protein expression levels, and they included a significant fraction (~45%) with similar expression levels in the cell line comparisons. These proteins created novel molecular signatures to differentiate the cancer cell lines studied here. Our results suggest that protein folding and stability measurements complement the current paradigm of expression level analyses for biomarker discovery and help elucidate the molecular basis of disease.

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Unfortunately, these studies have not been as useful as expected at identifying disease-related proteins that can be exploited for diagnostic and therapeutic purposes, presumably due to the indirect link between a protein’s expression level and its function. Investigated here is the use of thermodynamic stability measurements to probe a more biologically relevant dimension of the proteome. It has the potential to become a new strategy for disease state characterization and to help elucidate the molecular basis of the disease. This thesis outlines the use of two discovery based techniques and one validation based technique to study protein folding and stability changes associated with breast cancer.

The first part of this dissertation describes the application of a mass spectrometry-based technique, stable isotope labeling with amino acids in cell culture and stability of proteins from rates of oxidation (SILAC-SPROX), in a comparison of the MCF-7 versus BT-474 breast cancer cell lines and a comparison of the MCF-7 versus MDA-MB-468 breast cancer cell lines. This work enabled ~1000 proteins to be assayed for breast cancer-related thermodynamic stability differences. The 242 and 445 protein hits identified with altered stabilities in these comparative analyses created distinct molecular markers to differentiate the three cell lines.

The second part of this dissertation describes the development of a SILAC-based limited proteolysis (SILAC-LiP) strategy. The applicability of the protocol was demonstrated in a proof-of-principle study using proteins from a yeast cell lysate and a ubiquitous ligand. The SILAC-LiP protocol was further applied in a comparison of the MCF-7 versus MCF-10A cell lines. This work identified ∼200 proteins with cell line dependent conformational changes, as determined by their differential susceptibility to proteolytic digestion using the nonspecific protease, proteinase K. The overlap between the SILAC-LiP hits reported here and the SILAC-SPROX hits previously identified in these same cell lines was relatively small (~20%). Thus, this work indicates that the SILAC-SPROX and SILAC-LiP techniques can be used together to provide complementary information on the disease states.

Furthermore, the protein hits identified in both the SILAC-SPROX and SILAC- LiP experiments included a large fraction (∼70%) with no significant expression level changes. This suggests protein folding and stability measurements can provide information about disease states that is orthogonal to that obtained in protein expression level analyses.

The last part of this dissertation focuses on the establishment of targeted mass spectrometry-based validation assays for the protein biomarker candidates with altered thermodynamic stabilities identified in the SILAC-SPROX experiments. Application of the PAB-SPROX protocol on the MCF-7 cell lysate enabled reproducible identification and quantitation of a subset of prioritized target peptides.

Over the past two decades, a toolbox of mass spectrometry-based proteomic methods has been developed that enables the conformational properties of proteins and protein-ligand complexes to be probed in complex biological mixtures, from cell lysates to whole cells. The focus of this dissertation is the extension of these methodologies to the study of protein-gas and protein-metal interactions, an area of limited application. The goals of this work are two-fold. The first is to improve current mass spectrometry-based proteomic methods that measure protein folding stability, which is accomplished by the development of a chemo-selection strategy for proteolysis procedures and a “one-pot” approach that increases statistical significance while decreasing experiment costs. The second goal of this work is the application of these methodologies and others to the study of protein-gas and protein-metal interactions in complex biological mixtures (i.e., cell lysates), in which insights could be gained about gas and metal biological activities by surveying their interactions within a proteome. The first part of this dissertation describes in more detail the development and application of a semitryptic peptide enrichment strategy for proteolysis procedures (STEPP) that enables the isolation of information-rich semitryptic peptides. With the STEPP protocol, the number of semitryptic peptides increased by 5- to 10-fold and the amount of structural information was maximized in limited proteolysis experiments. The combination of the pulse proteolysis technique with a novel “one-pot” approach for data acquisition and analysis (one-pot STEPP-PP), resulted in false positive rates reaching close to zero (i.e., 0.09%) for a proof-of-principle drug target identification experiment for cyclosporine A and a yeast lysate. Described in the second part of this dissertation is the application of the improved proteolysis methodologies and others to multiple studies of protein-gas and protein-metal interactions on the proteomic scale. First, the development and application of protein stability measurements to the study of protein-gas interactions, specifically protein-xenon interactions, is described. A sample preparation protocol that was conducive to protein-gas binding studies is developed and validated against a known xenon-binding protein, metmyoglobin. Ultimately, this sample preparation protocol was employed in large-scale, proteome-wide SPROX and limited proteolysis experiments to identify xenon-interacting proteins in a yeast lysate. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known. Our survey of the proteome revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes and revealed correlations between the mechanisms by which ATP and Xe target proteins. Next, the application of one-pot STEPP-PP is described in the context of two research areas, both related to identifying the protein targets of metal-associated cell death processes. First described is the utilization of this technique in combination with protein expression level analysis to identify bacterial protein targets of copper delivered by small molecule ionophores. The protein folding stability and expression level profiles generated in this work enabled the effects of ionophore vs. copper to be distinguished and revealed copper-driven stability changes in proteins from processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (by 3-fold) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies. The second application of the one-pot STEPP-PP methodology is to the study of Fe- and Zn-mediated sensitization to erastin-induced ferroptotic cell death. Our approach enabled differential protein expression and protein folding stability measurements to be made on RCC4 cells exposed to excess iron and zinc along with the ferroptosis-inducing molecule erastin. Of the protein targets identified, a few have known ties to pathways involved in ferroptotic cell death, while others have not been previously linked with ferroptosis. Future work aims at assaying the potential metal binding properties of these proteins, to connect them to their metal-enhancing ferroptosis effects. The final research area described in this dissertation is the development and application of a novel metal-induced protein precipitation (MiPP) approach which exploits the protein precipitation properties of metals to study proteins that are susceptible to metal overload. Total protein precipitation as a function of metal concentration was assayed across various proteomes (bacterial, fungal, and mammalian) and metals (copper, zinc, iron, etc). Copper-induced protein precipitation was measured within E. coli and C. albicans proteomes by coupling precipitation curves with a bottom-up proteomics readout. Proteome-wide precipitation studies revealed a wide distribution of copper precipitation midpoints for the identified proteins within these species. A fundamental understanding of the biophysical basis of susceptibility or tolerance to metal precipitation can potentially be garnered through more in-depth analysis of the proteins that fall significantly outside the average precipitation midpoint of each proteome.

The detection and quantitation of protein-ligand binding interactions is important not only for understanding biological functions but also for the characterization of novel protein ligands. Because protein ligands can range from small molecules to other proteins, general techniques that can detect and quantitate the many classes of protein-ligand interactions are especially attractive. Additionally, the ability to detect and quantify protein-ligand interactions in complex biological mixtures would more accurately represent the protein-ligand interactions that occur in vivo, where differential protein expression and protein complexes can significantly affect a protein's ability to bind to a ligand of interest.

The work in this dissertation is focused on the development of new methodologies for the detection and measurement of protein-ligand interactions in complex mixtures using multiplex analyses. Methodologies for two types of multiplexed analyses of protein-ligand binding interactions are investigated here. The first type of multiplex analysis involves characterizing the binding of one protein target to many potential ligands, and the second type involves characterizing the binding of one ligand to many proteins. The described methodologies are derived from the SUPREX (stability of unpurified proteins from rates of H/D exchange) and SPROX (stability of proteins from rates of oxidation) techniques, which are chemical modification strategies that measure thermodynamic stabilities of proteins using a relationship between a protein's folding equilibrium and the extent of chemical modification. These two techniques were utilized in the development and application of several different experimental strategies designed to multiplex the analysis of protein-ligand interactions.

The first strategy that was developed involved a pooled compound approach for making SUPREX-based measurements of multiple ligands binding to a target protein. Screening rates of 6 s/ligand were demonstrated in a high-throughput screening project that involved the screening of two chemical libraries against human cyclophilin A (CypA), a protein commonly overexpressed in types of cancer. This study identified eight novel ligands to CypA with micromolar dissociation constants. Second, an affinity-based protein purification strategy was developed for the detection and quantitation of specific protein-ligand binding interactions in the context of complex protein mixtures. It involved performing SPROX in cell lysates and selecting the protein of interest using immunoprecipitation or affinity tag purification. A third strategy developed here involved a SPROX-based stable isotope labeling method for measuring protein-ligand interactions in multi-protein mixtures. This strategy was used in a proof-of-principle experiment designed to detect and quantify the indirect binding between yeast cyclophilin and calcineurin in a multi-component protein mixture. Finally, a quantitative proteomics platform was developed for the detection and quantitation of protein-ligand binding interactions on the proteomic scale. The platform was used to profile interactions of the proteins in a yeast cell lysate to several ligands, including the bioactive small molecules resveratrol and manassantin A, the cofactor nicotinamide adenine dinucleotide (NAD+), and two proteins, phosphoglycerate kinase (Pgk1) and pyruvate kinase (Pyk1). The above approaches should have broad application for use as discovery tools in the development of new therapeutic agents.