This thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an... Show moreThis thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an understudied modification—UFM1. While the first chapter describes the attempt to introduce an unnatural amino acid into proteins to enable covalent substrate capture, the subsequent chapter reports on a unique cascading activity-based probe capable of being relayed through the enzyme cascade. The repertoire of activity-based probes was later expanded to include the Ubiquitin-like modifiers SUMO and UFM1. Additionally, the enigmatic role of UFM1 was deciphered by adapting a proteomics method previously used for SUMO, to uncover UFM1-modifed substrates. This approach enabled the dissection of a relevant pathway governed by UFMylation—ribosomal function during SRP-mediated protein translocation. Show less