PROJECT SUMMARY Post-translational modifications (PTMs) involve the addition of chemical groups to proteins, modulating the structures or functions of the modified proteins to coordinate cellular processes. ADP-ribosylation, the addition of adenosine diphosphate ribose to proteins, exists in both a monomeric (MARylation) and polymeric (PARylation) form, allowing for more complex regulation. The balance between the synthesis and degradation of ADP-ribosylation forms is fundamental for regulating cellular processes. Disruption of this balance leads to human diseases like cancer and neurodegeneration. However, much remains to be learned about the respective contributions of MARylation and PARylation to the regulation of physiological processes and disease pathogenesis. Mass spectrometry (MS)-based proteomics methods are the go-to techniques for investigating PTMs in healthy and disease states. Previously published proteomics methods studying ADP-ribosylation have been successful in identifying modification sites on amino acids of varied chemical properties. However, all of these methods fail to identify the ADP-ribosylation form associated with each site. This proposal aims to develop a MS-based proteomics method to identify the site and form of ADP-ribosylation on a proteome-wide level. To preserve information about the form of ADP-ribosylation at modification sites, my method will either (a) keep ADP- ribosylation intact during enrichment or (b) determine whether the amino acid is MARylated or PARylated. With a tool to distinguish between the ADP-ribosylation forms at each site, we can identify potential functional differences of MARylated and PARylated proteins in healthy and disease states. Ultimately, this workflow will provide valuable insights into the biological functions of the different forms of ADP-ribosylation in physiological and disease states.