Quantifying amyloid fibrils in protein mixtures via infrared attenuated-total-reflection spectroscopy
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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.
KeywordsAmyloid fibrils Protein-mixture analysis Mid-infrared Spectral deconvolution Fourier-transform infrared spectroscopy (FTIR) Attenuated total reflection (ATR)
Assistance of A. Wilk and X. Wang (Institute for Analytical and Bioanalytical Chemistry, Univ. Ulm) during initial IR-ATR studies is greatly acknowledged. We are grateful for a graduate student fellowship provided by the International Graduate School in Molecular Medicine at the University of Ulm. This study was in part supported by the BMBF (Federal Ministry of Education and Research, Germany), the Competence Net Neurodegenerative Dementias (project: FTLDc), the JNPD Networks for Standardization of Biomarkers (BiomarkAPD, Sophia), the EU (project: NADINE), the Foundation of the State of Baden-Württemberg, the Boehringer Ingelheim Ulm University BioCenter (BIU), and the State of Baden-Württemberg Junior Professor Programme.
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