Development and Application of Mass Spectrometry-based Strategies for Proteomic Evaluations of the Thermodynamics and Kinetics of Protein Folding

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2021

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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.

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Cabrera, Aurora Faustina (2021). Development and Application of Mass Spectrometry-based Strategies for Proteomic Evaluations of the Thermodynamics and Kinetics of Protein Folding. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/24425.

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