## 3 Results and Discussion
### Angel's Blouse, Blue Layer (Sampled Point 3)  
Under the surface of the most recent painting, a blue layer was revealed (Figure 1, Sampled point 3). Minute quantities of the blue surface were removed and subjected to lipid extraction. The MALDI-MS spectrum in positive mode exhibited the presence of peaks at _m/z_ 732.8 and 760.8 recognized as phosphatidylcholines from egg yolk [43; 44; 45] most likely employed as a paint binder. MALDI-MS/MS spectra of precursor ions at _m/z_ 732.8 and 760.8 as protonated adducts were respectively recognized as PC (32:1) and PC (34:1). Their fragmentation pattern appears typical for the class of PC which are easily identified through the diagnostic phosphocholine head-related ion at _m/z_ 184 being the base peak when working in positive ionization mode. Concerning the precursor ion at _m/z_ 760.8 shown in Figure 6, the appearance of a very low abundance ion at _m/z_ 227.1 could correspond to [FA(14:1)+H]\({}^{+}\), thus suggesting the occurrence of a PC (14:1-20:0) considering that the sum composition should be PC(34:1). The presence of PC suggests the use of egg-based binder for this painting layer since phosphatidychines typically compose the lipid fraction of egg yolk [33; 46; 47]. The use of this binder is corroborated by the ATR-FTIR spectrum acquired on the sample (Figure 7). In this case, the main signals at 3340, 2916, 2849, 1734, 1711, 1643, and 1541 cm\({}^{-1}\) suggested the presence of a mixed binder of lipidic and proteinaceous components (such as egg yolk). In particular, the bands at 1734 and 1711 cm\({}^{-1}\) are consistent with the presence of aged egg yolk, imputable to aldehydes and acids from triglycerides and/or imide bonds by oxidation of protein amidic bonds [48; 49]. The spectral features at 1643 and 1541 cm\({}^{-1}\) are compatible respectively with amide I and amide II stretching, though the higher intensity of the latter can be related to the contribution of calcium and lead carboxylates [49; 50]. Calcium carboxylates could be responsible also for the shoulder at 1576 cm\({}^{-1}\). The presence of basic lead carbonate can be inferred by a shoulder at 3535 cm\({}^{-1}\) and the peaks at 1044 and 680 cm\({}^{-1}\), whereas the signals at 1406 and 874 cm\({}^{-1}\) are diagnostic for the use of CaCO\({}_{3}\). Moreover, it is evident the characteristic band of CN stretching at 2087 cm\({}^{-1}\), suggesting that Prussian blue is present as a pigment. Notably, the peak at about 1400 cm\({}^{-1}\) of blue sampled areas was assigned to both basic lead carbonate and calcite (CaCO\({}_{3}\)), even if its intensity is uneven in these two cases suggesting a stratigraphic differentiation.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Experimental** & **Theoretical** & **Sequence** & **Identified Protein** \\ _m/z_ & _m/z_ & **Sequence** & **Identified Protein** \\ \hline
1560.67 & 1560.73 & (K)STGISVFPGPGSPGR(G) + 4 ox & Collagen alpha-1(I) \\
1562.79 & 1562.79 & (R)GDKGETGEQGDGK(G) + 1 ox & Collagen alpha-1(I) \\
1562.80 & 1562.83 & (K)GAAGLGPVGAGAGGLPGKR(G) + 3 ox & Collagen alpha-2(I) \\  & 1586.74 & (K)GNSGEPGAPGSKGDTGAK(G) & Collagen alpha-1(I) \\
1586.74 & 1586.70 & (R)GETGPAGPSGAPGAPGAGSR(G) + 4 ox & Collagen alpha-1(III) \\
1648.79 & 1648.78 & (K)AGEDGHPGKGPRGPGR(G) + 2 ox & Collagen alpha-2(I) \\
1759.79 & 1759.82 & (K)GEPGPGPGPQGAPGPAGEGK(R) & Collagen alpha-1(II) \\
1832.80 & 1832.81 & (R)GPPGPGPGPGLAGPPGSGR(E) + 3 ox & Collagen alpha-1(I) \\
1922.94 & 1922.93 & (R)GERGGPGPCESGAAGPTGPGSGR(G) + 1 ox & Collagen alpha-2(I) \\  & 1922.86 & (K)GDBGGPGPGPGAGPGPGRGR + 5 ox & Collagen alpha-1(III) \\  & 1975.99 & (K)GEDGRCTGFLAG