Structural Differences in Aβ Amyloid Protofibrils and Fibrils Mapped by Hydrogen Exchange – Mass Spectrometry with On-line Proteolytic Fragmentation

We report here structural differences between Aβ(1-40) protofibrils and mature amyloid fibrils associated with Alzheimerʹs disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Aβ(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 °C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35–40 and the N-terminal segment 1–19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20–34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the β-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal ∼ten residues appear to be unstructured in both protofibrils and fibrils. The 20–30 segment of Aβ(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Aβ peptide.