# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 3 Results
### Secondary Structure Estimation for Gluten and Zein  
In the present study, the secondary structure of gluten was studied after hydration and mixing for 5 min. The measurement and data analysis were conducted based on the method described and tested on the reference proteins. For FTIR spectra of gluten and zein, a wider range (1500-1700 cm\({}^{-1}\)) has been selected to perform peak fitting in order to do a proper baseline correction. However, the amide II band has not been subjected to the quantitative analysis because of its less protein conformational sensitivity compared with amide I [6]. Nevertheless, the peaks at 1540, 1545, and 1551 cm\({}^{-1}\) are usually assigned to \(\alpha\)-helical structure, while the peaks at 1520 to 1525 cm\({}^{-1}\) are assigned to \(\beta\)-sheet secondary structures [45].  
The curve fitted spectra obtained from three replicates of hydrated gluten samples show good reproducibility (Figures S2-1-S2-10). The relative secondary structure contents of hydrated gluten were estimated from peak fitting of both FTIR and FT-Raman results. The percentage of \(\alpha\)-helix, \(\beta\)-sheet,  
Figure 5: Curve fitted amide I region (1600–1700 cm\({}^{-1}\)) in FT-Raman spectra of the reference proteins (**a**) immunoglobulin G, (**b**) concanavalin A, (**c**) trypsin, and (**d**) lysozyme; \(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coil areas are distinguished with the following patterns ( ), ( ), ( ), ( ), and ( -  
\(\beta\)-turn, and random coil structures are \(36\pm 1\), \(42\pm 1\), \(22\pm 0\), and \(0\pm 0\%\), respectively, as obtained from FTIR analysis. This data was consistent with the FT-Raman results that indicate the presence of \(36\pm 5\), \(48\pm 6\), \(22\pm 7\), and \(0\pm 0\%\)\(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coil structure, respectively. The FT-Raman spectra of gluten (Figure 6) shows 10 major bands related to secondary structure. The bands at 1632, 1641, 1673, and 1680 cm\({}^{-1}\) are assigned to \(\beta\)-sheet secondary structures, while those centered at 1664, 1686, and 1693 cm\({}^{-1}\) are originating from \(\beta\)-turn secondary structures. The \(\alpha\)-helix bands were centered at 1649 and 1655 cm\({}^{-1}\). The three low frequency bands near 1604 and 1609 cm\({}^{-1}\) do not contribute to the secondary structure and are related to aromatic side chain of amino acids [46].  
The secondary structure of zein was also investigated after hydration and mixing with water for 5 min at 40 \({}^{\circ}\)C (Figure 7). The amount of \(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coil were \(34\pm 2\), \(42\pm 4\), and \(0\pm 0\%\), respectively, based on FTIR measurements. These data matched well with FT-Raman results showing \(36\pm 2\), \(45\pm 2\), \(18\pm 4\), and \(0\pm 0\%\) of \(\alpha\)-helix, \(\beta\)-sheet, \(\beta\)-turn, and random coil, respectively. The reduced chi-square, \(R^{2}\), and adjusted \(R^{2}\) of gluten and zein peak fittings were calculated to assess the goodness of fit. The \(R^{2}\) and adjusted-\(R^{2}\) values were \(>0.99\) for all peak fittings indicating that the fitted values are close to the actual data points (Tables S2-1-S2-10). The chi-square value was reduced to less than 1E-6 and the fit was completely converged in all curve-fittings.  
Figure 6: Curve fitted amide I and II regions in (**a**) FTIR and (**b**) FT-Raman spectra of hydrated gluten; \(\alpha\)-helix, \(\beta\)-sheet, and \(\beta\)-turn areas are filled with the following patterns ( ), ( ), respectively.  
Figure 7: Curve fitted amide I and II regions in (**a**) FTIR and (**b**) FT-Raman spectra of hydrated zein; \(\alpha\)-helix, \(\beta\)-sheet, and \(\beta\)-turn areas are filled with the following patterns ( ), ( ), respectively.