# A Comparison of PCA-LDA and PLS-DA Techniques for Classification of Vibrational Spectra
## 3 Results and Discussion  
The normalized simulated spectra of control-like and exposed-like types were independently averaged in order to obtain mean spectra, which are shown in Figure 1a,b, respectively. As expected, the difference between control and exposed mean spectra, shown in Figure 1c, is characterized by large positive peaks centred at 785, 830, 1090 and 1580 cm\({}^{-1}\), corresponding to the spectral peaks whose intensity has been decreased for the basic spectrum in order to simulate damage in exposed-like type spectrum. The other positive and negative peaks in Figure 1c are related to random intensity differences thataffect the mean spectra and they are particularly relevant for the largest intensity features, as those centred at 1450 cm\({}^{-1}\), 1660 cm\({}^{-1}\) and 1200-1400 cm\({}^{-1}\) spectral range. Similarly, the normalized mean Raman spectra of unexposed and proton-exposed MCF10A cells are plotted in Figure 2a,b, respectively. Such spectra are characterized by Raman peaks and spectral features related to the main cellular components, as nucleic acids (784, 1096, 1340, 1373, 1490 and 1578 cm\({}^{-1}\)), proteins (1003, 1032, 1128, 1207, 1260, 1340, 1615 and 1662 cm\({}^{-1}\)) and lipids (1128, 1300 and 1440 cm\({}^{-1}\)) [29]. The positive peaks in the difference spectrum in Figure 2c suggest that the main effect of radiation exposure on the Raman spectra consists in a relative decrease of nucleic acid components, as a consequence of larger exposure damage to DNA/RNA than to protein and lipid components [27]. A significant intensity decrease of the Raman peaks related to the phosphodiester bond (at 784 cm\({}^{-1}\)) and DNA bases ring modes (at 1574 cm\({}^{-1}\)) was also reported by [PERSON] et al. in proton irradiated calf thymus DNA [30]. Modification of the above Raman peak related to nucleic acids was also reported by [PERSON] et al. for cellular samples exposed to different types of ionizing radiation, such as proton and \(\gamma\)-rays [31]. Figure 1: Synthetic spectra modelling control (**a**) and exposed (**b**) samples. The mean spectra are reported by blue and red lines in (**a,b**), respectively. Standard deviation spectra are also reported as dashed lines and the grey area corresponds to spectral values between mean \(\pm\) standard deviation values. The spectral differences between mean values are reported in (**c**) and the wavenumber values of spectral features mainly contributing to such a difference are labelled for clarity. Furthermore, the infrared absorption spectra of MCF7 and MDA cells, shown in Figure 3a,b, respectively, are characterized by spectral peaks and bands related to nucleic acids (1082, 1117 and 1227 cm\({}^{-1}\)), proteins (1165, 1306, 1390, 1448, 1535 and 1637 cm\({}^{-1}\)) and lipids (1390, 1448 and 1736 cm\({}^{-1}\)). The difference spectrum in Figure 3c indicates that the two types of cells can be biochemically discriminated according to the larger relative amount of nucleic acid content in MCF7 cells with respect to MDA ones, as suggested by the spectral peak at 1082 and 1227 cm\({}^{-1}\), whereas the peaks at about 1535 and 1637 cm\({}^{-1}\) are due to a relative spectral shift of the amide II and I band for the two types of cell [28]. Both such results are in good agreement with those reported in the literature. In particular, both [PERSON] et al. [32] and [PERSON] et al. [33] found a larger relative amount of nucleic acids in the MCF7 cells with respect to MDA cells. As for the shifts of amide I and II bands, they might be connected with changes in the secondary protein structures occurring during the process of carcinogenesis [28]. Figure 2: Raman spectra of control (**a**) and proton-exposed (**b**) MCF10A cells. The mean spectra are reported by blue and red lines in (**a**,**b**), respectively. Standard deviation spectra are also reported as dashed lines and the grey area corresponds to spectral values between mean \(\pm\) standard deviation values.