"Computational and Chemical Proteomic Strategies for Deconvoluting Inhibitor and Drug Mode of Action"

The analysis of proteomes using mass spectrometry has enabled the direct measure of the abundance for thousands of protein gene products from a single sample. Small molecule covalent probes can survey the activity of these proteins and through competition assays identify their ligands. This activity-based protein profiling (ABPP) provides the means to probe and potentially drug protein targets that may be otherwise inaccessible. Given that the development of new covalent probes enables the study of different subsections of the proteome, advances in proteomic and computational strategies are needed to optimize coverage and utility of probe modified proteomes.
This dissertation uses three different chemical proteomics strategies to 1) identify the targets of an inhibitor, 2) profile tyrosines across the proteome and 3) screen fragment electrophiles across the proteome. In addition, we present a computational tool kit built for the analysis of chemical proteomics datasets. Chapter 1 provides background on proteomics with mass spectrometry, ABPP, and the relevant computational strategies. In Chapter 2 we identify that repurposed serotonin receptor antagonist ritanserin, is cytotoxic in small cell and non-small cell lung cancer cell lines. Based on this finding we used a kinase specific chemical probe to identify c-Raf as an important ritanserin target in these cell lines. Chapter 3 details the development of SuTEx probes as an in vitro and live cell tyrosine selective probe. We use this probe to identify tyrosines that readily react with the SuTEx scaffold and monitor changes in phosphorylation state of tyrosines. Chapter 4 presents a computational platform for analysis of probe modified site-specific mass spectrometry datasets. Using this platform, we profile several probes, competitive ABPP data, and perform an in silico screen of covalent fragment electrophiles. Chapter 5 summarizes the findings and discusses future direction for these strategies.