## 3 Results and Discussion  
### Analysis of Infrared Spectra from Control and Exposed Cells  
In Figure 1, the average FT-IR spectra of control and irradiated cells are reported. As is evident, the experimental approach adopted and the preliminary data treatment allow to obtain good quality spectra, fully comparable with those collected using different acquisition conditions (see [14]). The spectra here obtained show the presence of several bands related to the vibrational modes of the biologic molecules of cells constituents (lipids, proteins, DNA, etc.). In the panel A and B, the average spectra obtained for the high wavenumber region (HWR) from 3600 to 2600 cm\({}^{-1}\) and for the fingerprint region (1800-900 cm\({}^{-1}\)) are, respectively, reported. As expected, the spectra show bands that are due to the contribution of proteins, lipids, and carbohydrates. Figure 2A shows the results of deconvolution procedure in the HWR for control samples. In particular, the bands in the range of 3200-3500 cm\({}^{-1}\) are due to the amide A (-N-H) stretching motion of peptide backbones of proteins amino acids and O-H stretching of carbohydrate polysaccharides, while the band at \(\approx\)3170 cm\({}^{-1}\) is mainly assigned to -NH\({}_{3}\) asymmetric stretching of free amino acids. The two peaks at \(\approx\)2960 cm\({}^{-1}\) and \(\approx\)2890 cm\({}^{-1}\) are related, respectively, to the asymmetric and symmetric stretching of the methyl groups (-CH\({}_{3}\)) given by cellular proteins and lipids contribution. The structures at \(\approx\)2920 cm\({}^{-1}\) and \(\approx\)2850 cm\({}^{-1}\) are, respectively, assigned to the asymmetric and symmetric stretching of the methylene groups of membrane lipids (-CH\({}_{2}\)). In Figure 2B, the deconvolution of the fingerprint region for control samples is reported and different peaks that are related to proteins and nucleic acids are evident. The two peaks at \(\approx\)1650 cm\({}^{-1}\) and \(\approx\)1540 cm\({}^{-1}\) are mainly due to the amide I (C=O and C-N) and amide II (N-H and C-N), respectively. The band at \(\approx\)1450 cm\({}^{-1}\) is attributed to symmetric and asymmetric bending of the methylene and methyl groups (-CH\({}_{2}\) and -CH\({}_{3}\)) and to -CH\({}_{2}\) scissoring of proteins and lipids, and the feature at 1400 cm\({}^{-1}\) is due to COO\({}^{-1}\) group asymmetric stretching of proteins. The asymmetric and symmetric -PO\({}_{2}\)\({}^{-}\) stretching vibrations of the phosphodiester nucleic acid backbone are associated with the two bands at \(\approx\)1230 cm\({}^{-1}\) and \(\approx\)1085 cm\({}^{-1}\), respectively, with a contribution from C-O-P stretching of protein and lipids. Figure 1: Comparison of average spectra for control and proton irradiated MCF-10A cells (**A**) in the high wavenumber region (3600–2600 cm\({}^{-1}\)) and (**B**) in the fingerprint region (1800–900 cm\({}^{-1}\)) at different proton doses (0.5, 2, 4 Gy); data are presented as Mean \(\pm\) SD. For each dose three slides were examined. On each slide, at least three regions were investigated, and three spectra were obtained for each position. A total number of at least 27 spectra was obtained. In Table 2, the assignments for all the resolved peaks are shown [2, 4, 5, 6, 35, 44]. In addition, the analysis of second derivative spectra (data not shown) of control cells allowed the identification of other contributions. These are also reported in Table 2 in bold characters and they are related to Amide III (1291 cm\({}^{-1}\)), C-H ring bending (1207 cm\({}^{-1}\)), sugar -phosphate backbone vibration (1171 cm\({}^{-1}\)), ribose C-O stretching (1143 cm\({}^{-1}\)) and P-O-C symmetric stretching (1100 cm\({}^{-1}\)) [14]. In Table 3, the position of peaks for samples exposed to different doses are reported. The shifts, in terms of wavenumber, of peak positions with respect to those found for the control samples are also reported; shifts higher than the spectral resolution of our experimental apparatus are indicated in bold character.