Mass-Spectrometry-Based GEE Footprinting Characterizes Kinetic Mechanisms and Sites of Conformational Change in...

Understanding the dynamics of Aβ aggregation is critical for elucidating Alzheimer's disease (AD) progression. This study extends our previous work on Aβ42 using fast photochemical oxidation of proteins (FPOP) and pulsed hydrogen-deuterium exchange and introduces mass spectrometry (MS)-based glycine ethyl ester (GEE) footprinting, combined with kinetic modeling, to characterize Aβ42 conformational changes and elucidate polymer populations along its aggregation pathways. We investigated Aβ42 conformational changes by analyzing three distinct peptide regions generated by Lys-N digestion, revealing three different views of the aggregation behaviors. The middle and C-terminal regions are identified as primary aggregation sites; in contrast, the N-terminal peptide exhibited only minor changes in GEE modification, supporting its limited involvement in intermolecular interactions during aggregation. Amino-acid-level analysis provided higher spatial resolution: D1 underwent relatively constant footprinting throughout aggregation, whereas E3/D7, E22, and D23 showed more substantial decreases in the level of modification, underscoring their critical roles in aggregation. By integrating these findings with kinetic modeling, we identified four predominant polymeric populations involved in Aβ1-42 aggregation. This study reports, for the first time, a stable, specific, and slow chemical footprinting approach to characterizing Aβ1-42 aggregation, offering new insights into Aβ1-42 polymerization dynamics and enhancing our understanding of its role in AD pathology. The solvent accessibility features of the six acidic amino acids and the C terminus calculated from the final, fibril state structure of Aβ42 are consistent with the footprinting results.