High-Throughput Screening of Amyloid Inhibitors via Covalent-Labeling Mass Spectrometry
Year: 2025
Type: article
Abstract: Amyloid fibril formation by proteins is implicated in numerous human diseases, yet few treatments exist in part due to the lack of robust screening methods for amyloid inhibitors. Here, we demonstrate a novel mass spectrometry (MS) assay for high-throughput screening of amyloid inhibitors, based on measuring the extent of protein labeling during protein aggregation. Amyloid formation decreases covalent labeling (CL) extents, while the presence of an inhibitor restores the extent of labeling, providing a means of identifying inhibitors. Using two different labeling reagents, α,β-unsaturated carbonyl (ABUC) and diethylpyrocarbonate (DEPC), and insulin and β2-microglobulin (β2m) as model amyloid proteins, we show that the CL-MS assay can probe protein amyloid formation and its inhibition by a wide range of compounds, with validation achieved by comparisons to traditional fluorescence and light scattering techniques. In proof-of-concept screens, several new inhibitors are identified for both proteins and further verified for their ability to fully prevent aggregation. Overall, our CL-MS assay offers fewer false positives than conventional methods and is compatible with high-throughput screening, achieving rates of >10 compounds per minute using matrix-assisted laser desorption/ionization (MALDI)-MS as a readout.
Source: Analytical Chemistry
Institution: University of Massachusetts Amherst
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W4411467147