On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy

Abstract

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.

Pathophysiological Response to SARS-CoV-2 Infection Detected by Infrared Spectroscopy Enables Rapid and Robust Saliva Screening for COVID-19

“…In addition, seven points of varying significance (p < 0.06-0.01) correlated with the significant peaks from the COVID.POS FU.POS vs. COVID.NEG comparison: right shift of amide I (1688/1658 cm −1 ); decreased aliphatic/RNA (1430 cm −1 ); decreased δCH 3 − bending (1373 cm −1 ); decreased ν s PO 2 − RNA (1124 cm −1 ), ν s PO 2 − , symmetric, and C-O ν ribose (1071 cm −1 ); decreased νC 4 -OH − glucose (1016 cm −1 ). The right shifting amide I peak in COVID.POS FU.POS , compared to both COVID.NEG and COVID.POS FU.NEG , is in agreement with residual misfolded amyloid protein fibrils and elevated IgA in COVID-19 patient saliva (Figure S5) [9,26,27].…”

Infrared spectroscopy enables rapid, robust, portable COVID-19 saliva screening based on pathophysiological response to SARS-CoV-2

“…In addition, 7 points of varying significance (p < 0.06-0.01) correlated with the significant peaks from COVID.POS FU.POS vs COVID.NEG comparison: right shift of Amide I (1688/1658 cm -1 ), decreased aliphatic/RNA (1430 cm -1 ), decreased δCH 3 - bending (1373 cm -1 ), decreased v s PO 2 - RNA (1124 cm -1 ), v s PO 2 − , symmetric and C-O ν ribose (1071 cm -1 ), and decreased v C 4 -OH - glucose (1016 cm -1 ). The right shifting Amide I peak in COVID.POS FU.POS compared to both COVID.NEG and COVID.POS FU.NEG is in agreement with residual misfolded amyloid protein fibrils and elevated IgA in COVID-19 patient saliva (S5 Fig)(4, 19, 20).…”

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli

“…This indicates the presence of long and rigid amyloid fibrils [ 36 , 56 ]. For the R2 fragment, a broad band located at about 1645 cm −1 in the Amide I, which is characteristic of disordered proteins [ 57 ], could be observed. After 30 days of incubation at 37 °C, we did not notice any significant changes in all studied ATR-FTIR spectra in the range of 1725–1590 cm −1 ( Figure 4 B).…”