Mass-Spectrometry-Based GEE Footprinting Characterizes Kinetic Mechanisms and Sites of Conformational Change in Amyloid β 1–42 Aggregation

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.