## 3 Results and Discussion
The band at \(\approx\)1660 cm\({}^{-1}\) is due to \(\alpha\)-helix secondary structures, while the random structures and \(\beta\)-turns contribute to the bands at \(\approx\)1650 and in the 1670-1685 cm\({}^{-1}\) range, respectively  
Ten subcomponents related to parallel \(\beta\)-sheet (1629, 1637 cm\({}^{-1}\)), anti-parallel \(\beta\)-sheet (1609, 1619 cm\({}^{-1}\)), \(\alpha\)-helix (1653, 1660 cm\({}^{-1}\)), \(\beta\)-turn (1665, 1675, 1689 cm\({}^{-1}\)), and unordered structure (1644 cm\({}^{-1}\)) were used to analyze the Amide I band of control and irradiated samples of MCF-10A cells exposed to different proton doses and fixed at different times (see panel III in Figure S1 for details). The main components of the Amide I band are related to the \(\beta\)-sheet and \(\alpha\)-helix. The area ratios between the areas of the different  
Figure 2: Results of the deconvolution of the average control spectra (**a**) in the 3600–2600 cm\({}^{-1}\) and (**b**) in the 1800–900 cm\({}^{-1}\) ranges. In the panels, the attributions of the main peaks are also reported. components of the Amide I band compared to that of the whole Amide I band do not point to statistically significant changes for the different radiation doses and different fixation times here examined, apart from a change in the \(\alpha\)-helix component for t\({}_{88}\) samples exposed to higher doses (see Table S2 and Figure S1). Similar behavior has already been observed in our previous work using the same cells exposed to the same proton doses: in that case, only cells fixed immediately after exposure were considered [24]. This behavior differs from that observed for X-ray-exposed neuroblastoma cells when examined by \(\mu\)-FT and Raman spectroscopy [32; 49]. As already reported in the previously mentioned paper, since Amide I band analysis did not indicate any significant difference in the secondary structures of cell proteins, additional information was obtained by deconvolving the Amide III spectral range (see panel IV in Figure S1 for details). In fact, the Amide III region can also be used to characterize the secondary protein structure [38; 39]. The absorption signal in the 1290-1330 cm\({}^{-1}\) range is generally attributed to the \(\alpha\)-helix structure, whereas random coils and \(\beta\)-sheets are related to signals around 1240-1270 cm\({}^{-1}\) and 1180-1240 cm\({}^{-1}\), respectively [32; 39; 50]. In Figure 3 and in Table S3, the results of the deconvolution procedure are reported. It is evident that the main contribution to the Amide III band can be related to the \(\beta\)-sheet component, which shows significant changes for some doses at different fixation times. The other relevant component is represented by the random coil, which also demonstrates some significant variations. The behavior of the different Amide III components is analogous to what was observed in the previous work, which considered only cell samples fixed immediately after exposure. The modifications in Amide III subcomponents are probably related to changes in the contributions of enzymes involved in DNA repair (see [23; 51] and references therein), as already reported for the Amide I band [32]. However, the interpretation of changes present in the Amide III region should be considered with caution since contributions from other cellular components could be present in a different manner compared to the cases reported in Refs. [38; 39; 50] in regard to protein samples. Figure 3: Changes in secondary protein structure contributions to Amide III band (1350–1180 cm\({}^{-1}\)) with dose for cells fixed immediately and 24 h, 48 h, and 72 h after irradiation; the ratios between the secondary structure peak areas and the area of the whole Amide III peak are reported as Mean \(\pm\) SD. Asterisks indicate when a significant difference from the corresponding control value occurred at \(p\leq 0.05\).