A label-free method to quantify early-stage amyloid aggregation under flow via intrinsic phenylalanine fluorescence

TL;DR

This paper introduces a label-free flow-based fluorescence method using phenylalanine signals to quantify early amyloid aggregation, including small oligomers, under near-physiological conditions, addressing limitations of traditional assays.

Contribution

The study develops a novel flow dispersion analysis technique leveraging intrinsic phenylalanine fluorescence to monitor early amyloid aggregation stages, especially for peptides with low aromatic content.

Findings

Successfully quantified oligomer size evolution from 2 to 10 nm.

Validated method on tryptophan-free, fast-fibrillating peptides.

Extended analysis to small, heterogeneous oligomers under physiological conditions.

Abstract

The aggregation of amyloid-forming peptides is a dynamic, complex process that underlies their diverse biological activities, from physiological functions to disease-associated dysfunctions. While the structure of fibrillar end-products is well-characterized for most amyloids, the heterogeneous and often transient oligomers, likely key in cytotoxicity, remain poorly investigated, especially for peptides with low-yield aromatic residues. Here, by exploiting and developing flow induced dispersion analysis in both peak and front modes, we demonstrate that intrinsic phenylalanine fluorescence can be harnessed to quantify the conversion of diffusing monomers into non-diffusing oligomers and fibrils. We further characterize low-molecular-weight oligomers, and their size evolution from 2 to 10 nm over time. Importantly, we validate the robustness of our approach using two tryptophan-free and fast-fibrillating amyloid peptides, PSM$\alpha$3 and hIAPP, known for their key roles in S. aureus virulence and type 2 diabetes respectively. Our results overcome the limitations of traditional biochemical and biophysical amyloid assays by extending analysis from large oligomers and fibrils to small heterogeneous oligomers, under near-physiological conditions. This study thus offers a new analytical framework, thereby filling a critical gap in amyloid research, to probe the early stages of aggregation, key in the design of alternative therapeutics for amyloid-diseases.