The “amyloid hypothesis” posits that Amyloid β (Aβ) misfolds, oligomerizes to soluble species, and thereby contributes to neurodegenerative Alzheimer’s disease (AD). Recent support for the hypothesis comes from a new antibody, Lecanemab, that targets Aβ and its aggregation and significantly reduces clinical decline. Still, the aggregation is not understood, leaving a weak foundation for building new therapeutics. In the transition from monomer to fibril, Aβ exists in soluble, putatively neurotoxic species in mixtures of varied sizes and morphologies. Complicating matters further is the unknown molecular role of apolipoprotein E (ApoE), a family of partially disordered proteins including an isoform, ApoE4, that is one of few biomarkers for AD. The aggregate mixture and the interactions of Aβ with apoE4 and with lipids constituting cell membranes are too complex for many structural characterization techniques. Protein footprinting coupled with mass spectrometry, however, can provide a measure of solvent accessibility, local dynamics and structure, hydrogen bonding, sites of ligand- binding, and oligomerization, with spatial resolution at the peptide and amino-acid residue levels. Considerable preliminary data establish that this technique can provide structural details not obtainable by other methods. This footprinting technique has not yet been systematically or rigorously applied to characterize the soluble intermediates formed in amyloidogenic protein aggregation, and this is our goal. We will chemically footprint Aβ and ApoE alone or together by using both pulsed hydrogen/deuterium exchange (HDX) and irreversible modification by highly reactive species (free radicals, carbenes, carbocations). For the latter, we will utilize the “Fast Photochemical Oxidation of Proteins” or “FPOP” platform, which we invented. Both HDX and FPOP are used in a differential mode where changes in structure are determined as a function of aggregation time or increasing media complexity. Utilization of optimized reagents will be coupled with separation and kinetic modeling to reduce heterogeneity and analyze the aggregation pathways and the varied structures or morphologies of the oligomers. We will then apply these optimized footprinting approaches in complex media containing lipids, small molecules, and other proteins including antibodies to reveal how aggregation changes upon perturbation with these substances. We will be guided by powerful Density Functional Theory that can probe small molecule/Aβ interactions Once the footprinting and data processing pass rigorous validation, we will apply them to Aβ and ApoE in a system of reprogrammed neurons that recapitulate the pathophysiology of AD patients. This system succeeds where pluripotent stem cells and mouse models do not because they don’t carry age information, and age correlates strongly with AD. The proposed research will bring new understanding of Aβ aggregation and of ApoE interactions with regional and amino ...