# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 4 Discussion
These bands were not included in the quantitative analysis of the FT-Raman results; only the area under the peaks present between 1620 and 1699 cm\({}^{-1}\) region were studied for the FT-Raman analysis of the protein secondary structure. In addition to the structural analysis of the reference proteins, the secondary structure of gluten and zein proteins was also investigated by both FTIR and FT-Raman spectroscopy using the same analysis method optimized for the reference proteins. Similar to the procedure for reference proteins, the pure liquid water spectrum was subtracted from the FTIR spectra of zein and gluten. Other approaches to correct for water for the hydrated states of gluten proteins have already been explored such as using spectra of H\({}_{2}\)O-D\({}_{2}\)O mixtures as the correcting spectra [18]. However, the bending frequency of HDO(Deuterium hydrogen oxide) coincides with the amide II band, which makes its analysis virtually impossible [1]. In addition, the change in the shape of the amide II band as a result of HDO signal can affect the peak fitting of the amide I since it can change the baseline of the spectrum. Therefore, the pure water spectrum was subtracted from the zein and gluten FTIR spectra in this study. Gluten proteins are typically classified into two major sub-groups: glutenins and gliadins [52]. The'monomeric' gliadins, which are imparting viscosity in a viscoelastic gluten network, are often described to be built as a three domain structure: a short N-terminal domain, a repetitive central domain rich in glutamine and proline, and a non-repetitive C-terminal domain rich in sulfur containing amino acids. According to previous reports, the N-and C-terminal domains are rich in \(\alpha\)-helix structures, while the central domain mainly adopts \(\beta\)-turn secondary structures in hydrated gluten [52; 53]. The glutenin macropolymers have a similar three domain structure. In hydrated gluten, the glutenin central domain comprises mainly \(\beta\)-turn secondary structures forming \(\beta\)-spiral super secondary structures [53]. The \(\beta\)-spirals are assumed to contribute to the intrinsic elasticity of a viscoelastic gluten network [54]. Based on the loop and train model proposed by [PERSON] [55], \(\beta\)-turn structures are present in the loop regions (water-protein interactions) while the intermolecular \(\beta\)-sheets are dominating the train regions (protein-protein interactions). It seems that the interchain \(\beta\)-sheet structures are formed at the expense of \(\beta\)-turns [18]. The intermolecular \(\beta\)-sheet structure showed two signature peaks at 1616 and 1623 cm\({}^{-1}\)[49], corresponding to 36% of the total amide I band. As mentioned earlier, this strong short wavenumber peak (1616 cm\({}^{-1}\)) is specifically due to the \(\beta\)-sheets with stronger hydrogen bonds [37; 49], and its presence shows the formation of high amount of intermolecular \(\beta\)-sheets upon hydration and mixing of gluten. The formation of intermolecular \(\beta\)-sheets has been also attributed to the association of high molecular weight (HMW) glutenin subunits [56]. Zein proteins lack the HMW glutenin subunit-type structures [57] which can explain the absence of 1616 cm\({}^{-1}\) hidden band in the zein FTIR spectra (Figure 7a) compared with gluten FTIR spectra (Figure 6a). Similarly, the band at 1620 cm\({}^{-1}\) in FT-Raman spectra is also assigned to intermolecular hydrogen-bonded \(\beta\)-sheets [58] which is more pronounced in hydrated gluten FT-Raman spectra (Figure 6b) compared with what is observed for hydrated zein (Figure 7b). In addition, the absorption band at 1636 cm\({}^{-1}\) is characteristic of amide groups involved in the extended \(\beta\)-sheets in the gluten network [59]. The appearance of this band has been attributed to kneading and stretching processes [59]. The gluten in its unhydrated state (9% moisture content) has been reported to contain 39% \(\beta\)-sheet, 30% random coil, 17% \(\alpha\)-helix, and 14% \(\beta\)-turn structure [18].