Leveraging the full potential of Raman spectroscopy for the identification of amyloid aggregation states

Abstract Background Raman spectroscopy is a label‐free method often used in the characterization of the beta‐amyloid (Aβ) aggregation process. This process involves peptide misfolding, which can be tracked with Raman microscopy in a non‐invasive manner, since the technique is sensitive for conformational changes in peptide structure. However, often only the spectral bands corresponding to β‐sheets and α‐helices are used for the evaluation of the aggregation state, leaving the remaining spectral information unused. The here presented evaluation aims to explore the full potential of Raman spectroscopy for the characterization of Aβ‐42 aggregates (monomers, oligomers, fibrils) in a comprehensive analysis approach, complemented by Thioflavin T (ThT) fluorescence measurements and atomic force microscopy (AFM). Method Aβ‐42 was reconstituted in diluted ammonia. were prepared by reconstituting peptide in diluted ammonia on ice and dilution with phosphate buffered saline (PBS). Oligomers and fibrils were obtained by incubation in MilliQ water or diluted hydrochloric acid for 24 hours at 4°C or 37°C, respectively. Samples were optionally further diluted for AFM, ThT fluorescence assays and . The Raman spectra were processed using Project 4 software (WITec GmbH, Ulm, Germany), and in‐house written Matlab scripts (The Mathworks, Inc., Natick, USA). Result Using Raman spectroscopy, as well as established methods such as AFM and ThT fluorescence, the size and aggregation state of monomeric, oligomeric and fibrillar Aβ‐42 could be confirmed and reproducibly obtained using standardized protocols. Corresponding Raman spectra of these Aβ‐42 aggregates display distinct differences not only in the β‐sheet and α‐helix spectral band, but in the whole spectrum, indicating especially changes in the orientation of tyrosine, phenylalanine, and aliphatic side chains. Conclusion Herein, the potential of Raman spectroscopy as a label‐free, conformation‐sensitive technique for the characterization of Aβ‐42 aggregates was explored by analyzing the full Raman spectrum. Clear differences between all aggregate types were identified in several spectral bands, unveiling detailed information in the Raman spectra that is often not included in the analysis of Aβ aggregates. Thus, this study provides a guideline for extracting comprehensive knowledge from spectroscopic data on peptide aggregation, potentially serving as a non‐destructive, quick addition or alternative to more established methods.