Quantitative approach to study secondary structure of proteins by FT-IR spectroscopy, using a model wheat gluten system

Amide I and Amide III vibrational modes are frequently used to study protein secondary structure with Fourier transform infrared (FT-IR) spectroscopy. However, for protein mixtures, neither the sole Amide I nor Amide III region provides sufficient information for structural quantitation because of overlapping peaks, especially in the Amide I region. Here, an improved quantitative approach is proposed to estimate secondary structure of protein systems using resolution enhancement and curve-fitting data processing techniques on a gluten model system to investigate structure-function relationships. Twelve different scenarios were prepared to assign bands in the Amide I region. Frequency ranges of 1660–1640 cm⁻¹ and 1665–1660 cm⁻¹ were found to highly contribute to variability in secondary structure contents of samples. Utilization of the Amide III region as a conducive tool to assign bands in the Amide I region led to a better differentiation of some secondary structural motifs and a more accurate quantitation of protein secondary structure. The study presents an understanding of FT-IR data analysis for a quick technique to assess secondary structures of protein mixtures.