Quantifying amyloid fibrils in protein mixtures via infrared attenuated-total-reflection spectroscopy

Abstract

Amyloid aggregation of proteins is usually associated with amyloid diseases. A distinct feature of protein aggregation is the increase of crossed β-sheet structures. Infrared attenuated-total-reflectance (IR-ATR) spectroscopy is a sensitive optical technique that has the potential to provide secondary structure characteristics of proteins even in complex biological samples. In this study we report the analysis of secondary structures of proteins, using the amide I band for the detection and quantification of amyloid fibrils in protein mixtures by use of IR-ATR techniques, at comparatively low sample concentrations. From the experimental results, an analytical model of the relationship between the IR spectra of protein mixtures and the individual mixture components was established using spectral-deconvolution procedures and curve-fitting methods. On the basis of this model, four ratios were shown to provide direct information on amyloid aggregated fibrils via the increase of crossed β-sheet structures in protein-mixture samples. In conclusion, this study confirms the utility of IR spectroscopy for analyzing protein mixtures and for identifying amyloid fibril information within such complex multi-component samples.

Mid‐infrared thin‐film diamond waveguides combined with tunable quantum cascade lasers for analyzing the secondary structure of proteins

“…On the other hand, γ‐globulin is rich in β‐sheet structures, and the amide I band is correspondingly more pronounced at approximately 1639 cm −1 . These results are in agreement with previous studies carried out on silicon ATR waveguides .…”

“…Protein spectra analyzed with the conventional FTIR spectrometer were subject to a similar treatment routine, as previously described . IR spectra were acquired and processed with Opus 6.5 software, which was also used to exclude water vapor and CO 2 interferences.…”

Advances in Mid-Infrared Spectroscopy for Chemical Analysis

“…For example, the aggregation state of α-synuclein or amyloid β (Aβ) peptides, which are both associated with neurodegenerative diseases such as Parkinson's or Alzheimer's, have been studied via MIR spectroscopy (110). Probing mechanistic details of misfolding and fibrillation (122,123), as well as the inhibition of these processes, along with potential environmental influences and stimulants has revealed band shifts characteristic of the amide I (1,600-1,700 cm −1 ) and amide II (1,500-1,600 cm −1 ) features of proteins, which may be assigned to α-helix or β-sheet conformations (124,125).…”

Recent advancements of EC-QCL based mid-IR transmission spectroscopy of proteins and application to analysis of bovine milk1

“…The sensitivity to individual secondary structure elements originates in differing patterns of hydrogen bonding, dipole-dipole interactions and geometric orientations in the α-helices, β-sheets, turns and random coil structures that induce different frequencies of the C=O vibrations [4]. It has been shown that based on the analysis of the amide I band, quantitation of individual proteins in a protein mixture can be performed [47].…”