DEVELOPMENT OF CLICK-CHEMISTRY BASED ENRICHMENT STRATEGIES TO CHARACTERIZE LIGAND BINDING AND METABOLISM

Abstract

The production and perception of biogenic small molecules (BSMs) form the basis of chemical communication between cells, organisms, and the environment. BSMs such as hormones, odors, and neurotransmitters are well known examples of compounds that allow us to sense our environment and are required for our growth and development. Therefore, understanding the history of BSMs from their biosynthesis to their perception will furnish a better understanding of biology. In order to characterize the BSM-ligand receptor binding necessary for their perception, platforms such as Activity (Affinity) Based Protein Profiling (A(f)BPP) have made use of crosslinking probes that are structurally similar to the BSM-ligand of interest. Upon binding and crosslinking to the target protein binding pocket, these probes can then facilitate the enrichment of the resulting probe-protein complex. Enzymatic digestion and MS/MS analysis of the enriched species can then help identify the target of the probe. A(f)BPP has been extraordinarily useful for identification and characterization of enzymes but the use of this technology to identify the likely targets of BSM perception (i.e., membrane-bound receptors) is very limited. On the other hand, metabolomic studies can shed light on the biosynthesis of BSMs. Through untargeted analytical platforms, researchers can discover novel metabolites, and therefore novel metabolic pathways. These analyses have been greatly enhanced by advances in analytical instrumentation, including the introduction high resolution mass spectroscopy (HRMS) to allow for more sensitive and accurate analysis of metabolic features. However, overwhelming datasets and the prioritization of compounds that can be accessed with current instrumentation biases metabolomic analysis towards compound classes that are abundant or have already been previously identified. This dissertation presents the development of two strategies that employ click-chemistry to enrich and then annotate either peptides from the binding pockets of receptor proteins, or the metabolic products of alkyne-labeled probes. In this way, the former strategy attempts to characterize the initial BSM-ligand binding event, while the latter strategy is used to discover novel metabolic pathways through the analysis of enriched species.