## 3 Results and Discussion
### Analysis of Amide I and Amide III Bands  
The analysis of the different peaks constituting the Amide I band can give accurate information on the secondary structure of proteins and their conformational changes due to different doses of ionizing radiation [6; 36; 37; 38]. The Amide I band (Figure 2C) can be considered as a convolution of the contributions due to the various secondary structures of proteins; the bands between 1620-1640 cm\({}^{-1}\) and \(\approx\)1690 cm\({}^{-1}\) can likely be attributed to \(\beta\)-sheet structures with the \(\approx\)1620 and 1690 cm\({}^{-1}\) band characteristic of the anti-parallel \(\beta\)-sheet structures. The band at \(\approx\)1660 cm\({}^{-1}\) is principally assigned to \(\alpha\)-helix secondary structures, while the random structures and \(\beta\)-turns make a contribution to the bands at \(\approx\)1650 and in the 1670-1685 cm\({}^{-1}\) range, respectively. In the present case, the analysis of the Amide I band of control samples and irradiated samples was performed using ten subcomponents related to parallel \(\beta\)-sheet (1629, 1637 cm\({}^{-1}\)), anti-parallel \(\beta\)-sheet (1609, 1619 cm\({}^{-1}\)), \(\alpha\)-helix (1653, 1660 cm\({}^{-1}\)), and \(\beta\)-turn (1665, 1675, 1689 cm\({}^{-1}\)); unordered structures are attributed to the band at 1644 cm\({}^{-1}\).  
In the present case, the ratios between the area of the different subcomponents of the Amide I band and the area of the entire Amide I band do not show statistically significant variations when the different proton doses are considered (data not shown). Since Amide I band analysis did not evidence any variation in the secondary structure of cell protein content, the deconvolution of Amide III region (Figure 2D) was performed in order to obtain additional information. In fact, also the Amide III region can offer interesting information about secondary protein structure [39]. The spectral band in 1290-1330 cm\({}^{-1}\) range is generally assigned to the \(\alpha\)-helix secondary structure. Assignments for random coil (1240-1270 cm\({}^{-1}\)) and \(\beta\)-sheets (1180-1240 cm\({}^{-1}\)) were also determined [39; 48]. In Figure 3, it is evident that the largest contribution to the Amide III band is due to the \(\beta\)-sheets component that does not show any significant changes for the different doses here investigated. Conversely, the \(\alpha\)-helix component shows a significant increase for the 2 and 4 Gy doses. The random coil contribution shows a large change for 0.5 Gy dose. Using Amide III band deconvolution changes in the protein secondary structure induced by proton irradiation has been noted. In Table 4 detailed information on the different subcomponent contributions to Amide III is reported. The Amide changes here discussed for proton irradiated cells had not so far been shown using \(\mu\)-FT-IR [12; 13; 14].  
\begin{table}
\begin{tabular}{c c c c c} \hline
**0 Gy** & **Assignment** & **0.5 Gy** & **2 Gy** & **4 Gy** \\ \hline
1304 & p, 1 & 1304 & 1303 (\(-\)1) & 1311 (+7) \\
1291 & p & 1286 (\(-\)5) & 1295 (+4) & 1287 (\(-\)4) \\
1227 & DNA, c & 1227 & 1231 (+4) & 1233 (+5) \\
1207 & DNA, RNA & 1203 (\(-\)4) & & 1202 (\(-\)5) \\
1171 & DNA & 1171 & 1171 & 1169 (\(-\)2) \\
1166 & 1 & 1166 & 1170 (+4) & 1167 (+1) \\
1143 & DNA & 1149 (+5) & & \\
1124 & DNA & 1124 & 1121 (\(-\)3) & 1116 (\(-\)8) \\
1100 & DNA & 1098 (\(-\)2) & & 1097 (\(-\)3) \\
1086 & DNA, p & 1086 & 1085 (\(-\)1) & 1084 (\(-\)2) \\
1058 & DNA, p & 1058 & 1051 (\(-\)7) & 1058 \\
958 & DNA, p & 965 (+7) & & 966 (+8) \\ \hline \end{tabular}
\end{table}
Table 3: _Cont._