# Mass Spectrometry-Based Protein Footprinting: A New Tool for Amyloid Protein Aggregation

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2024 · $748,641

## Abstract

The “amyloid hypothesis” posits that Amyloid β (Aβ) misfolds, oligomerizes to soluble species, and thereby
contributes to neurodegenerative Alzheimer’s disease (AD). Recent support for the hypothesis comes from a
new antibody, Lecanemab, that targets Aβ and its aggregation and significantly reduces clinical decline. Still, the
aggregation is not understood, leaving a weak foundation for building new therapeutics. In the transition from
monomer to fibril, Aβ exists in soluble, putatively neurotoxic species in mixtures of varied sizes and morphologies.
Complicating matters further is the unknown molecular role of apolipoprotein E (ApoE), a family of partially
disordered proteins including an isoform, ApoE4, that is one of few biomarkers for AD. The aggregate mixture
and the interactions of Aβ with apoE4 and with lipids constituting cell membranes are too complex for many
structural characterization techniques. Protein footprinting coupled with mass spectrometry, however, can
provide a measure of solvent accessibility, local dynamics and structure, hydrogen bonding, sites of ligand-
binding, and oligomerization, with spatial resolution at the peptide and amino-acid residue levels. Considerable
preliminary data establish that this technique can provide structural details not obtainable by other methods. This
footprinting technique has not yet been systematically or rigorously applied to characterize the soluble
intermediates formed in amyloidogenic protein aggregation, and this is our goal. We will chemically footprint Aβ
and ApoE alone or together by using both pulsed hydrogen/deuterium exchange (HDX) and irreversible
modification by highly reactive species (free radicals, carbenes, carbocations). For the latter, we will utilize the
“Fast Photochemical Oxidation of Proteins” or “FPOP” platform, which we invented. Both HDX and FPOP are
used in a differential mode where changes in structure are determined as a function of aggregation time or
increasing media complexity. Utilization of optimized reagents will be coupled with separation and kinetic
modeling to reduce heterogeneity and analyze the aggregation pathways and the varied structures or
morphologies of the oligomers. We will then apply these optimized footprinting approaches in complex media
containing lipids, small molecules, and other proteins including antibodies to reveal how aggregation changes
upon perturbation with these substances. We will be guided by powerful Density Functional Theory that can
probe small molecule/Aβ interactions Once the footprinting and data processing pass rigorous validation, we will
apply them to Aβ and ApoE in a system of reprogrammed neurons that recapitulate the pathophysiology of AD
patients. This system succeeds where pluripotent stem cells and mouse models do not because they don’t carry
age information, and age correlates strongly with AD. The proposed research will bring new understanding of
Aβ aggregation and of ApoE interactions with regional and amino ...

## Key facts

- **NIH application ID:** 10739659
- **Project number:** 1R01AG079283-01A1
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** MICHAEL L GROSS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $748,641
- **Award type:** 1
- **Project period:** 2024-05-15 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10739659

## Citation

> US National Institutes of Health, RePORTER application 10739659, Mass Spectrometry-Based Protein Footprinting: A New Tool for Amyloid Protein Aggregation (1R01AG079283-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10739659. Licensed CC0.

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