On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy
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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 interme...diates 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.
Keywords:
amyloid fibrillation / ATR FTIR / HEWL / oligomer intermediates / secondary structure perturbationSource:
Molecules, 2021, 26, 4, 970-Publisher:
- MDPI
Funding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200168 (University of Belgrade, Faculty of Chemistry) (RS-MESTD-inst-2020-200168)
Note:
- Supplementary material: https://cherry.chem.bg.ac.rs/handle/123456789/4391
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- Version of
https://doi.org/10.3390/molecules26040970 - Referenced by
https://cherry.chem.bg.ac.rs/handle/123456789/4391
DOI: 10.3390/molecules26040970
ISSN: 1420-3049
WoS: 000624154100001
Scopus: 2-s2.0-85102698528
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Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Milošević, Jelica AU - Prodanović, Radivoje AU - Polović, Natalija PY - 2021 UR - https://cherry.chem.bg.ac.rs/handle/123456789/4388 UR - https://www.mdpi.com/1420-3049/26/4/970 AB - 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. PB - MDPI T2 - Molecules T1 - On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy VL - 26 IS - 4 SP - 970 DO - 10.3390/molecules26040970 ER -
@article{ author = "Milošević, Jelica and Prodanović, Radivoje and Polović, Natalija", year = "2021", 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.", publisher = "MDPI", journal = "Molecules", title = "On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy", volume = "26", number = "4", pages = "970", doi = "10.3390/molecules26040970" }
Milošević, J., Prodanović, R.,& Polović, N.. (2021). On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy. in Molecules MDPI., 26(4), 970. https://doi.org/10.3390/molecules26040970
Milošević J, Prodanović R, Polović N. On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy. in Molecules. 2021;26(4):970. doi:10.3390/molecules26040970 .
Milošević, Jelica, Prodanović, Radivoje, Polović, Natalija, "On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy" in Molecules, 26, no. 4 (2021):970, https://doi.org/10.3390/molecules26040970 . .