Summary/Abstract Late Onset Alzheimer's disease (LOAD) is the most common form of age-related dementia. Currently, 5 million people in the US are afflicted with LOAD and with the aging population this number is expected to double in the next decades. There are no drugs to cure or halt the progression of LOAD. The two pathological hallmarks of LOAD are extracellular amyloid plaques formed by the insoluble A peptide and neurofibrillary tangles consisting of hyperphosphorylated Tau protein. To date, most clinical drug candidates have targeted A, but none have proven effective at ameliorating the symptoms of AD. New drug targets are needed to combat this increasingly common and devastating disease. The recent identification of LOAD risk factors has revealed an enrichment of proteins in the endosomal-lysosomal network (ELN). This corroborates decades of evidence that the disruption of the ELN is an early event in LOAD pathogenesis and indicates that the ELN contains potential drug targets. However, a lack of molecular characterization of ELN has prevented the discovery of suitable candidates. Once considered “undruggable”, protein-protein interactions (PPI) are emerging as attractive targets for drug development. Global analysis of PPI in the ELN has not been studied. We propose to use mass spectrometry to quantitate different biochemical characteristics ELN protein complexes and determine how LOAD induces alterations in PPI within the ELN. These experiments will be performed in human brain tissues (AD vs. age-matched controls) and in human AD and control IPSC derived neurons and organoids. Endogenous ELN targets will be immunoprecipitated with validated antibodies and quantitated between conditions to identify novel ELN interactors and disease-relevant drug targets. Quantitation will be performed using multiplexing isobaric labeling technology. The structure of the ELN complexes will also be resolved, and differences will be quantitated with using our Covalent Protein Painting method. Additionally, we will employ a novel application of the non-canonical amino acid, azidohomoalanine, to quantitate the stability of ELN protein complexes. Our proposal will produce three different quantitative measurements of the influence of LOAD pathogenesis on ELN protein complexes and will provide alternative drug development targets for the most common form of age-related dementia.