Unveiling Brain A beta Heterogeneity Through Targeted Proteomic Analysis

Amyloid beta (A beta) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Besides classic full-length peptides, biochemical analyses of brain deposits have revealed high degree of A beta heterogeneity likely resulting from the action of multiple proteolytic enzymes. This chapter describes a sequential extraction protocol allowing the differential fractionation of soluble and deposited A beta species taking advantage of their differential solubility properties. Soluble A beta is extracted by water-based buffers like phosphate-buffered saline-PBS-whereas pre-fibrillar and fibrillar deposits, usually poorly soluble in PBS, are extractable in detergent containing solutions or more stringent conditions as formic acid. The extraction procedure is followed by the biochemical identification of the extracted A beta species using Western blot and a targeted proteomic analysis which combines immunoprecipitation with MALDI-ToF mass spectrometry. This approach revealed the presence of numerous C- and N-terminal truncated A beta species in addition to A beta 1-40/42. Notably, the more soluble C-terminal cleaved fragments constitute a main part of PBS homogenates. On the contrary, N-terminal truncated species typically require more stringent conditions for the extraction in agreement with their lower solubility and enhanced aggregability. Detailed assessment of the molecular diversity of A beta species composing interstitial fluid and amyloid deposits at different disease stages, as well as the evaluation of the truncation profile during various pharmacologic approaches will provide a comprehensive understanding of the still undefined contribution of A beta truncations to AD pathogenesis and their potential as novel therapeutic targets.