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High-Throughput Screening of Amyloid Inhibitors via Covalent-Labeling Mass Spectrometry
Kanitin Khamnong1, Vanessa L Stahl2, Olivia N Izikson1
1Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States.
Analytical Chemistry
|June 19, 2025
Summary
A new mass spectrometry (MS) assay effectively screens for amyloid inhibitors by measuring protein labeling changes during aggregation. This covalent labeling MS (CL-MS) assay identifies potential treatments for amyloid diseases with high throughput and fewer false positives.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Drug Discovery
Background:
- Protein amyloid fibril formation is linked to various human diseases.
- Current treatments are limited due to a lack of effective screening methods for amyloid inhibitors.
- Robust assays are needed to identify compounds that can inhibit amyloid formation.
Purpose of the Study:
- To develop and validate a novel mass spectrometry (MS) based assay for high-throughput screening of amyloid inhibitors.
- To demonstrate the utility of the assay using insulin and beta2-microglobulin as model amyloid proteins.
- To identify novel inhibitors of amyloid formation.
Main Methods:
- A covalent labeling mass spectrometry (CL-MS) assay was developed.
- The assay measures changes in protein labeling extent during aggregation.
- Two labeling reagents (ABUC, DEPC) and two model proteins (insulin, β2m) were used.
- Assay performance was validated against fluorescence and light scattering techniques.
- Matrix-assisted laser desorption/ionization (MALDI)-MS was used as the readout for high-throughput screening.
Main Results:
- Amyloid formation was shown to decrease covalent labeling extents.
- Inhibitors restored the extent of protein labeling, enabling inhibitor identification.
- The CL-MS assay successfully probed amyloid formation and inhibition.
- Several new inhibitors for insulin and β2m were identified.
- The assay achieved screening rates exceeding 10 compounds per minute.
Conclusions:
- The developed CL-MS assay is a robust and high-throughput method for screening amyloid inhibitors.
- This assay offers advantages over conventional methods, including reduced false positives.
- The CL-MS assay has significant potential for accelerating the discovery of treatments for amyloid-related diseases.
