Amyloid β Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer's Disease

Numerous A beta proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated A beta proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent A beta-amyloidosis. Though the brain-derived A beta proteoform landscape is largely occupied by A beta 1-42, 25 different forms of A beta with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble A beta, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.