Large-Scale Analysis of Protein-Gas and Protein-Metal Interactions using Mass Spectrometry-Based Proteomic Methods

Abstract

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.