## 3 Results and Discussion  
In Figure 1, the average FT-IR spectra in the high-wavenumber range (HWR, from 3600 to 2600 cm\({}^{-1}\)) and fingerprint range (1800-900 cm\({}^{-1}\)) of unexposed and proton-beam-exposed cells are reported for several doses and for different fixation times. The spectra are characterized by a few overlapping peaks caused by the contribution of vibrational modes of the main cellular biomolecular constituents (lipids, proteins, DNA, and carbohydrates). The spectra show similar contributions from these components, with some differences related to the different doses and fixation times. It can be noticed that spectra collected for t\({}_{8}\) and t\({}_{72}\) samples are noisier than the spectra from cells fixed at shorter times. This behavior could be ascribed to effects due to delayed fixation times [42, 43] and to the presence of unrepaired cells, as discussed in Ref. [27], where a similar situation is described for Raman spectra obtained for proton-exposed MCF-10A cells. In Figure S1, the details of the deconvolution procedure in the different spectral ranges of interest are shown, and, in Figure 2, these results are summarized for control samples fixed immediately after the exposure for the HWR and fingerprint regions. In the two panels, the assignments of the main peaks are also reported [18; 19; 21; 24]. In Table S1, the spectral position of absorption peaks for exposed samples are reported, as well as their spectral shifts compared to those obtained for the control samples fixed at different times. Shifts larger than the spectral resolution of our apparatus are indicated in red bold character. In the HWR, some shifts in the OH stretching region (at 3492 and 3401 cm\({}^{-1}\)) are shown for the different doses and fixation times. In addition, the peak around 2880 cm\({}^{-1}\) due to CH\({}_{3}\) symmetric stretching and related to membrane lipids and proteins shows a shift larger than the spectral resolution, at 4 Gy for t\({}_{0}\) samples and at 0.5 Gy for t\({}_{72}\) samples. These changes in peak position are usually associated with changes in membrane fluidity [44]. Similar shifts have also been previously observed for X-ray-irradiated neuroblastoma cells [32] and in a previous paper on proton-exposed MCF-10A cells fixed just after exposure [24]. In the fingerprint region, numerous wavenumber shifts are visible, especially for cells fixed at 48 h and 72 h after exposure. In this region, the observed shifts are related to changes in DNA contributions (around 1300-1000 cm\({}^{-1}\)). In particular, the shift in the PO\({}_{2}\)\({}^{-}\) DNA stretching modes (located around 1226 and 1285 cm\({}^{-1}\)) can be due to modifications in the DNA conformation from the B-DNA to the A-DNA form, as indicated by [PERSON] et al. [45] by means of Raman spectroscopy. These variations in the DNA region were further investigated using the previously mentioned ratiometric approach. Amide I band analysis is generally adopted in order to obtain detailed information about protein secondary structures and their conformational modifications due to external agents [38; 39; 40; 46] and to different doses of ionizing radiation [18; 24; 32; 47; 48]. Various secondary structures can contribute to the Amide I signal; the contributions between 1620-1640 cm\({}^{-1}\) and \(\approx\)1690 cm\({}^{-1}\) are generally attributed to \(\beta\)-sheet structures, with the \(\approx\)1620 and 1690 cm\({}^{-1}\) bands characteristic of anti-parallel \(\beta\)-sheet structures.