Characterization of Abeta aggregates using hydrogen/deuterium exchange mass spectrometry

The work presented in this dissertation aimed to localize the hydrogen-bonding interaction sites within Abeta aggregates associated with Alzheimer's disease using hydrogen/deuterium exchange mass spectrometry (HDX-MS) coupled with proteolysis. Toward this end, an on-line digestion system utilizing a triaxial electrospray probe as the front end to MS was developed. Using this approach, regions of Abeta(1-40) fibrils and protofibrils that are protected from HDX have been identified. The HDX data show that the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to exchange in both fibrils and protofibrils, while the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The HDX data of fibrils match a model in which residues 11-23 and 28-36 are involved in highly protected structures, while those of protofibrils fit a model in which residues 14-20 and 31-36 are contained in highly protected structures. The N-terminal ∼10 residues and the C-terminal ∼4 residues appear to be unstructured in both fibrils and protofibrils. The 20-30 segment of Abeta(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 Abeta peptide.; The on-line digestion system has been extended successfully with three pepsin-like enzymes (protease type XIII, protease type XVIII and endothiapepsin). Preliminary proteolysis results show that individual or combinational use of the enzymes leads to production of more fragments of Abeta(1-40) (thus potentially more detailed structural information) than using pepsin alone.; The oxidation of Abeta samples was encountered in the course of these studies. It was found that gradual corrosion of a stainless steel electrospray emitter under conditions of normal use can generate surface irregularities that sustain electrical discharge. The increased emission current can affect the electrochemical reactions associated with the spray process, including oxidation of methionine to methionine sulfoxide in Abeta(1-40) peptides. The resultant mass shift and reduced sensitivity can adversely affect HDX experiments. These effects can be avoided by adding a redox buffer or (preferably) by re-polishing the emitter.