One of biology's central conundrums is how the cell regulates complex biochemical reactions in time and space. Cells have solved this problem by producing compartments, namely organelles, comprised of distinct chemical environments. Emerging evidence suggests that multiple "membrane-less" organelles within the cell assemble via phase separation and behave like droplets within an aqueous environment. In vivo and in vitro, such phase separation is primarily driven by intrinsically disordered domains or proteins (IDPs). The structural plasticity of IDPs allows them to adopt various structural conformations, generating multiple, weakly adhesive, inter- and intramolecular interactions. Unfortunately, this conformation flexibility comes at the cost of an increased risk of protein jamming or aggregation. Evidence suggests that imbalances between the thermodynamic drive to undergo phase separation and the established opposing aggregation control machinery could lead to disease. For example, amyotrophic lateral sclerosis (ALS) is believed to arise from aberrant phase separation of transactive response (TAR) element DNA binding protein of 43 kDa (TDP-43). TDP-43 is an essential RNA binding protein that plays a fundamental role in mRNA splicing, transport, and stability. Despite having a vast knowledge of the individual domains of TDP-43, there has been relatively little study of the full-length protein that is found in biologically relevant phase separation. In particular, the structural and sequence-specific basis of TDP-43 phase separation is not well understood and remains elusive. Our lab has a very successful track-record with incorporating hydrogen/deuterium exchange mass spectrometry (HXMS) to solve emerging biological questions. The overall objective of this proposal is to utilize HXMS to understand the structural and molecular cues that trigger TDP-43 phase separation. I hypothesize that there are small, 3-5 amino acid long, regions within the N-terminal, first RNA-recognition motif, and C-terminal domains of TDP-43 that are required for phase separation. Additionally, this proposal will look at linking the structural dynamics of ALS-disease variants with their increased propensity to phase separate. I hypothesize that certain mutations perturb the structure of the C-terminal domain of TDP-43, producing larger or additional contact points for self-association, leading to an increased degree of phase separation. My proposed experiments will provide a basic understanding of how full-length and variant TDP-43 behaves in solution and lay the foundation for the structure- based development of efficient inhibitors.