Proteoforms in Proteinopathies
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Description: Distinct amyloid-beta (Aβ) conformers such as peptides, oligomers (AβOs), and fibrils have long been targets studied for the cause, diagnosis, and treatment of Alzheimer’s disease (AD). Spatiotemporal spreading of AβOs is theorized to underly AD progression; however, because of significant polydispersity, no consensus has been reached into which AβO structural elements or size distribution lead to potent neurotoxicity. Evidence suggests Aβ exists in diverse modified proteoforms or is associated with cofactors (e.g., metals). The diversification of Aβ monomers may contribute to different rates of oligomerization, resulting in distinct AβOs. We propose a workflow that includes both denatured and native-state Aβ immunoprecipitation (IP) from brain tissues to study the compositional makeup of these monomers and how they vary in a manner associated with stages and brain regions during AD. The denatured state assays use top-down LC-MS/MS for the qualitative discovery of Aβ proteoforms followed by their quantitation via a novel proteoform-specific reaction monitoring (PfRM) assay. The approach has allowed for identifying and consistent relative quantitation of a panel of proteoforms across brain tissue samples with molecular level specificity. Additionally, label-free quantitation (LFQ) applied on these proteoforms allows for a deeper interpretation of the data around enzymatic processing of Amyloid Precursor Protein (APP) that allows for the formation of Aβ truncation products.