# Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins
## 3 Results
### Secondary Structure Estimation for Standard Proteins
\begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{2}{c}{**Amide I Position (cm\({}^{-1}\))**} & \multicolumn{2}{c}{**Assignment**} \\ \hline \multirow{5}{*}{**FTIR**} & 1610–1627 & \multicolumn{2}{c}{Intermolecular \(\beta\)-sheet} \\  & 1628–1642 & \(\beta\)-sheet \\  & 1643–1650 & Random coil \\  & 1650–1659 & \(\alpha\)-helix and Gln sidechain \\  & 1660–1699 & \(\beta\)-turn \\ \hline \multirow{5}{*}{**Raman**} & 1620–1648 & \(\beta\)-sheet \\  & 1649–1660 & \(\alpha\)-helix \\ \cline{1-1}  & 1660–1665 & Random coil \\ \cline{1-1}  & 1665–1680 & \(\beta\)-sheet \\ \cline{1-1}  & 1658–1680 & \(\beta\)-turn \\ \cline{1-1}  & 1680–1699 & \(\beta\)-turn \\ \hline \hline \end{tabular}  
* Data adapted from refs [5, 38, 39].  
\end{table} 
Table 1: Secondary structure assignments of amide I band components in proteins *. Figure 2: Infrared spectrum in the amide I region of the aqueous solution of concanavalin A. The upper and lower figures represent the curve fitted, and the second derivative spectrum, respectively. infrared spectra of lysozyme showed a relatively narrow amide I band with a maximum near 1654 cm\({}^{-1}\), which is the characteristic of proteins with a large content of \(\alpha\)-helical secondary structure [19; 23] (Figure 3). After curve fitting, lysozyme had a distinct \(\alpha\)-helical component of 46% at the 1654 cm\({}^{-1}\) band position. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Protein**} & \multicolumn{4}{c}{**Secondary Structure (\%)**} & \multirow{2}{*}{**Method**} \\ \cline{2-2} \cline{4-6}  & \(\alpha\)-Helix & \(\beta\)-Sheet & \(\beta\)-Turn & \multicolumn{1}{c}{**Random Coil**} \\ \hline \multirow{3}{*}{Immunoglobulin G} & 7 \(\pm\) 1 & 67 \(\pm\) 2 & 19 \(\pm\) 3 & 7 \(\pm\) 1 & FTIR \\  & 9 & 64 & 21 & 6 & FT-Raman \\  & 3 & 67 & 18 & 12 & X-ray [42] \\ \hline \multirow{3}{*}{Concanavalin A} & 9 \(\pm\) 1 & 57 \(\pm\) 1 & 22 \(\pm\) 1 & 12 \(\pm\) 0 & FTIR \\  & 8 & 59 & 23 & 10 & FT-Raman \\  & 3 & 60 & 22 & 15 & X-ray [42] \\  & 8–20 & 41–67 & 8–15 & 6–36 & CD [7; 8] \\ \hline \multirow{3}{*}{Trypsin} & 16 \(\pm\) 2 & 46 \(\pm\) 3 & 29 \(\pm\) 2 & 9 \(\pm\) 3 & FTIR \\  & 14 & 44 & 29 & 12 & FT-Raman \\  & 9 & 56 & 24 & 11 & X-ray [42] \\ \hline \multirow{3}{*}{Lysozyme} & 46 \(\pm\) 3 & 21 \(\pm\) 1 & 26 \(\pm\) 1 & 8 \(\pm\) 0 & FTIR \\  & 42 & 27 & 27 & 4 & FT-Raman \\  & 45 & 19 & 23 & 13 & X-ray [42] \\  & 45 & 21 & 26 & 8 & CD [8] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Relative amount of protein secondary structures determined by FTIR and FT-Raman peak fitting and comparison with X-ray and circular dichroism (CD) studies. Figure 3: Curve fitted amide I regions (1600–1700 cm\({}^{-1}\)) in FTIR 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 (**–  
FT-Raman spectra of the same reference proteins were collected to complement the FTIR data. For this purpose, the 20% w/v protein aqueous solutions were prepared and were allowed to stir for an hour at room temperature before measurement. The protein solutions were prepared at higher concentration for FT-Raman since FT-Raman spectra are characterized by a lower signal to noise ratio compared with FTIR. The FT-Raman spectra of standard proteins were then subjected to 15-point Savitsky-Golay smoothing to increase the signal to noise ratio. The original FT-Raman spectra of standard proteins in aqueous solutions show that water does not heavily distort the Raman spectra (Figure 4). Water only shows a very weak Raman signal in the amide I region, making subtraction of the water spectrum redundant. Thus, the FT-Raman spectra of the proteins were not subjected to water subtraction. The curve fitted plots from the FT-Raman profiles show the presence of different components in the amide I region (Figure 5).