Project Summary Several human proteins are known to form amyloid fibrils, and these fibrils are associated with devastating diseases, including Alzheimer's, Parkinson's, type II diabetes, and dialysis- related amyloidosis (DRA). The protein β-2-microglobulin (β2m) forms amyloid fibrils in the joints of patients undergoing hemodialysis, leading to DRA. While protein amyloid formation has been extensively studied, molecular-level information about the early stages of amyloid formation is only beginning to be revealed for a few proteins. This information, though, is critical for the rational development of therapeutics against amyloid diseases, particularly for DRA that has no treatment. Our group has been developing and applying new mass spectrometry (MS) methods to reveal structural and oligomeric changes that β2m undergoes before forming amyloid fibrils. Such structural information is very challenging to obtain using traditional biophysical methods, and the structural insight from our new methods has led to exciting discoveries about the first steps of protein amyloid formation. We are beginning to utilize this insight to find inhibitors of DRA. The research in this MIRA project seeks to (i) develop new methods that will deepen our mechanistic insight into the first steps of β2m amyloid formation and (ii) leverage this new mechanistic information to discover robust inhibitors of β2m amyloid formation. These goals will be accomplished by creating new MS methods that rely on covalent labeling, ion mobility, and computational modeling. These new approaches will allow us to probe the energy landscape of the structural switch that initiates β2m amyloid formation, providing unprecedented quantitative insight into the factors that cause this normally stable protein to become amyloidogenic. We will also explore new methods to characterize the on- and off-pathway isomeric oligomers that are present during the early stages of β2m amyloid formation, allowing us to reveal the specific protein-protein interactions that lead to amyloids. Altogether, the structural insights that we obtain will then be utilized to discover new inhibitors of β2m amyloid formation. Specific outcomes of this research will be new biophysical tools to study protein amyloid formation and the identification of inhibitors that could lead to therapeutics against DRA. The universality of the techniques developed in this work will also make them applicable to other amyloid systems and diseases.