APPLICATIONS OF CHEMICAL PROTEOMICS IN DEFINING PRENYLATED PROTEOMES AND DISCOVERING NOVEL PRENYLATED PROTEINS

Abstract

Since its development as a strategy to study biological systems, click chemistry has found its widespread use as a chemical tool in the discovery of post-translationally modified (PTM) proteins. In particular, the number of identified and validated lipid-modified proteins has dramatically increased over the last decade owing to the versatility and ease of use of this technique. Among these lipid PTMs, protein prenylation has benefited in uncovering new protein substrates, leading to the characterization and understanding of the functional role of this type of modification. This dissertation describes the development of a mass spectrometry-based chemical proteomics strategy that heavily relies on click chemistry to identify prenylated proteins in various systems, which allows for defining the prenylated proteomes and discovering novel prenylated proteins. The first chapter details a comprehensive literature review on the impact of click chemistry in revolutionizing the field of protein lipidation. Chapter 2 describes the development of chemical proteomics using probe analogue of isoprenoids to define the set of prenylated proteins in the malaria parasite, Plasmodium falciparum. Upon moving to eukaryotic systems, optimization of the probe and statin (enhancer of probe incorporation) treatments is described in Chapter 3. Our chemical proteomics approach was then successfully employed in various mammalian cell lines, enabling the validation of known and discovery of novel prenylated proteins. Moreover, the similarities and differences in brain-related cell lines were determined. Finally, an unusually prenylated protein, ALDH9A1, was discovered from these proteomic studies and tools in chemical and molecular biology were used to characterize and understand its occurrence. This dissertation highlights the advantages of using chemical proteomics in studying protein prenylation and contributes to the growing picture of protein lipid modifications.