Project Description To perform their multitude of functions, proteins often bring—distal in sequence—residues together and fold into reproducible higher-order structures (HOS). Ensuring the integrity of this orientation or intentionally modulating it through post-translational modification (PTM) are vital processes that are known to be perturbed in cancers, neurodegenerative, and cardiovascular diseases. Characterizing how PTM alters protein HOS can be employed to identify disease state and inform and accelerate the design of new targeted therapeutic interventions. Thus, to improve diagnostic capabilities, drug development, and provide insight into the fundamental mechanisms driving cellular function and disease, developing techniques capable of characterizing all the various PTM-modulated HOSs that exist for each protein is vitally important. The sensitivity, high-throughput nature, and low sample requirements of mass spectrometry (MS) has increasingly led to its adoption as a tool to measure protein HOS and PTM. Unfortunately, the most employed workflow, bottom-up proteomics, demands proteolytic digestion of the analyte and thus, it is not possible to distinguish the proteoform from which any single peptide is derived. Therefore, bottom-up workflows report the average conformation of all existing proteoforms and the average occupancy of all PTMs. Thus, to precisely assess the PTM-based regulation of protein function, over the next five years, we propose to develop new technologies that couple top-down proteomics, ion mobility spectrometry, chemical derivatization, and collision-induced unfolding. These new methods will be developed using well characterized model protein systems prior to application to more complex biological systems. The combination of these techniques will offer significant insight into these critical regulatory processes. In addition to these biological insights, we anticipate that this research proposal will generate a series of MS-based analytical techniques and an assortment of chemical reagents that will be applicable to a wide range of protein systems.