Evaluation of Amyloid-β Aggregation by Fourier Transform Infrared Spectrophotometer (FTIR)

Importance of the Topic

Amyloid-β fibril formation is central to the pathology of Alzheimer’s and related neurodegenerative disorders. Tracking how these peptides assemble into β sheet–rich aggregates provides critical insights into disease mechanisms and supports the development of aggregation inhibitors.

Objectives and Overview of the Study

This work aims to monitor the structural evolution of human amyloid-β(1–40) over time by examining changes in its amide I infrared band. Time-resolved measurements reveal the shift from antiparallel to parallel β sheet arrangements during aggregation.

Methodology

Amyloid-β was prepared at 10 μM in ultrapure water and incubated at 25 °C. Samples were collected at 0.5, 1, 2, 3, 24, 48, and 120 hours, dried on an ATR crystal, and analyzed by FTIR. Second derivative spectra identified characteristic peaks, and Lorentzian curve fitting quantified the proportion of secondary structure elements.

Used Instrumentation

• Shimadzu IRTracer-100 Fourier transform infrared spectrophotometer
• MicromATR accessory with a nine-reflection ATR crystal (Czitek, LLC)
• DLATGS detector with a dry air purge to reduce water vapor interference
• Measurement settings: 4 cm⁻¹ resolution, 100 scans accumulation, 4× zero filling, Sqr-Triangle apodization

Main Results and Discussion

• The primary amide I peak shifted from 1626 cm⁻¹ at 0.5 h to 1630 cm⁻¹ at 120 h, indicating structural rearrangement.
• Second derivative spectra showed decreasing intensity of antiparallel β sheet markers at ~1696 and ~1626 cm⁻¹ and the emergence of the parallel β sheet band at ~1630 cm⁻¹.
• Curve fitting demonstrated a drop in the ratio of high-wavenumber (1700–1690 cm⁻¹) to low-wavenumber (1645–1615 cm⁻¹) β sheet peak areas from 0.2836 to 0.1860, confirming a time-dependent shift toward parallel β sheet formation.

Benefits and Practical Applications of the Method

FTIR-ATR offers rapid, label-free detection of protein secondary structure changes with minimal sample preparation. It is ideal for screening aggregation inhibitors, ensuring peptide batch quality, and comparative analysis of disease-relevant variants.

Future Trends and Potential Applications

Emerging approaches include in situ ATR-FTIR under controlled temperature or pH to capture real-time aggregation kinetics, integration with microfluidic or high-throughput platforms for drug discovery, and advanced data processing methods, such as machine learning, to enhance spectral deconvolution.

Conclusion

Time-resolved ATR-FTIR effectively reveals the conversion from antiparallel to parallel β sheet structures during amyloid-β aggregation. This technique provides a robust platform for studying peptide assembly pathways and evaluating therapeutic strategies.

References

1. Sean D. Moran and Martin T. Zanni. J. Phys. Chem. Lett. 2014, 5, 1984–1993.
2. Mitsuo Tasumi. Infrared Spectroscopy – Fundamentals and Recent Techniques. ST Japan INC.
3. Jilie Kong and Shaoning Yu. Acta Biochim. Biophys. Sin. 2007, 39(8), 549–559.
4. Youcef Fezoui. Amyloid: Int. J. Chin. Invest. 2000, 7, 166–178.
5. Rabia Sarroukh et al. Biochim. Biophys. Acta 1828 (2013), 2328–2338.
6. Jennifer Kovacs-Nolan. J. Agric. Food Chem. 2005, 53, 8421–8431.