Elizaveta 'Liza' Shestoperova (Strieter group): Ubiquitination is a protein posttranslational modification in eukaryotic cells where a single ubiquitin or polymeric ubiquitin (polyUb) chains are attached to a protein substrate. Different ubiquitin chain types modifying a protein substrate adopt distinct topologies, often resulting in diverse functional outcomes in cells. The lack of universal methods for the analytical characterization of the conjugated polyUb chain topology makes it challenging to assess the relationships between the ubiquitin chain and its function. In my work, we evaluate the capabilities of multimodal mass spectrometry-based approaches, including ion mobility-mass spectrometry (IM-MS), to analyze various free or anchored-to-protein substrate isomeric polyUb chains. Previously, we have demonstrated that IM-MS can differentiate and identify multiple Ub oligomers. To strengthen IM-MS's capabilities for Ub isomer analysis, we coupled IM-MS with collision-induced unfolding (CIU). We demonstrated that Ub isomers obtain unique CIU fingerprints that can be used as their signature. Taking this advantage, we could access the enzymatic selectivity of different deubiquitinating enzymes towards Ub conjugates, opening the potential for real-world applications of this technique in ubiquitin-proteasome system (UPS) biological studies.