## 3 Results and Discussion
### Angel's Wing, Blue Layer (Sampled Point 1)
ATR-FTIR characterization allowed the identification of the blue pigment giving additional information about the organic component present in this investigated layer (Figure 3). The sharp signal falling at 2087 cm\({}^{-1}\) is uniquely assigned to the stretching vibration of the CN group of Prussian Blue [29], which was first introduced at the beginning of the XVIII century. Moreover, signals ascribable to other inorganic components such as basic lead carbonate (at 3508, 1398, 680 cm\({}^{-1}\)) and calcite (vibrations at 873, 712 cm\({}^{-1}\)) as well as to sulfate groups (1076 cm\({}^{-1}\)) could be identified [30]. The presence of sicctive oil as a binder was corroborated by the intense peaks at 1728, 2920, and 2850 cm\({}^{-1}\): while the first one was assigned to ester C = O stretching in the triacylglycerols of the drying oil, the other two to asymmetric and symmetric methylene stretching of the alkyl chains. The spectral features at 1237 and 1150 cm\({}^{-1}\) are compatible with the C-O-C stretching, also typical of the triglyceride ester linkages found in oils [31]. Oxidation of lipids is suggested by the wide band around 3440 cm\({}^{-1}\), assigned to -OH stretching [32]. Furthermore, the presence of proteinaceous components is supported by small peaks at 1650 and 1542 cm\({}^{-1}\), corresponding to amides I and II vibrational modes [32]. In the latter case, the contribution of metal soaps (e.g., lead carboxylates) cannot be ruled out [32]. Figure 3: ATR-FTIR spectrum of blue layer (Angel’s wing). The main peaks cited in the discussion are labelled. Figure 2: MALDI-MS spectra comparison between protein fraction from angel blue wing (**A**, above) and paint replica made of egg white mixed to CaCO\({}_{3}\) (**B**, below). K stands for Keratin and E for Enzyme due to auto-proteolytic digestion. After IR analysis, the sample was extracted by the Bligh and Dyer protocol (see Experimental Section), and the lipid fraction was analyzed by MALDI-MS. The spectrum examination of Figure 4A suggests the existence of a sicactive oil since some of the most common signals of diacylglycerols (DAGs) at _m_/z 603.5 and 617.5 and triacylglycerols (TAGs) at _m_/z 851.6, 881.6, 895.7, and 953.7 [33,34] were observed as sodiated adducts. It is known that the pigments combined with a binding medium speed up the conversion of lipids into oxidized products being the degree of oxidation directly related to the light exposure and/or aging time. The spectrum of Figure 4A is dominated by signals spaced 14 u (CH\({}_{2}\) units) or 16 u (oxygen unit) apart, mainly due to oxidation reactions or by-products generation containing shorter fatty acyl chains likely formed through the \(\beta\)-cleavage mechanism [33]. TAGs or DAGs with highly unsaturated fatty acyl chains such as C18:2 and C18:3 were not found due to their faster degradation rate. Indeed, the oxidation rate drastically depends on the number of unsaturations per chain, increasing over ten times for each double bond [35]. Tandem MS analysis of the precursor ion at _m_/z 881.6, reported in Figure 4B, confirmed that the most abundant product ions were those in which the neutral loss of fatty acids is implied. Therefore, peak signals at _m_/z 599.4 and _m_/z 625.4 correspond to [M-FA(18:1)+Na]\({}^{+}\) and [M-FA(16:0)+Na]\({}^{+}\), thus suggesting the occurrence of C18:1 and C16:0 chains in the examined TAG species. The ion at _m_/z 577.4 was interpreted as [M-FA(18:1)+H]\({}^{+}\) while the peak signal at _m_/z 607.5 was recognized as [M-FA(16:0)-H\({}_{2}\)O+Na]\({}^{+}\). At the low _m_/z range, a small ion at _m_/z 283.2 due to protonated fatty acid C18:1 (most likely oleic acid) was detected. The compound was thus recognized as the sodium adduct of TAG(52:2) composed of two oleic fatty acyl chains (O, 18:1) and one palmitic fatty acyl chain (P, 16:0), usually abbreviated as POO [36,37].