## 3 Results and Discussion
### Angel's Wing, Blue Layer (Sampled Point 1)  
The analyzed point on the blue layer of musician angel's left wing revealed a complex stratigraphy under the most recent pale layer unveiling three different colored layers: the most ancient one is gilded bole, the second is blue, and the third appears green (Figure 1, Sampled point 1). On the blue layer area, we performed an extensive investigation by applying both the micro- and the non-invasive approaches. The extracted peptides by the non-invasive strategy were examined by MALDI-MS (Figure 2A) and the _m_/z values were inserted in the database for peptide mass fingerprinting (PMF) search. The spectra were also manually compared to those of the replicas prepared as previously described [20; 21] and some chosen peptides were subjected to MS/MS analysis as a confirmation. For a direct comparison, Figure 2B shows the MALDI mass spectrum of the painting replica composed of egg white mixed with calcium carbonate, on which the same protocol (gel loaded with double enzyme) was carried out. As mentioned above, some critical matters to be managed in ancient statue samples are represented by the likely color stratigraphy, the interventions, retouching, and piece substitution with a continuous human manipulation that drastically reduced proteome identification alongside the well-known aging processesoften enhanced by entrapped pigments. The MALDI mass spectrum of dual-enzyme digest (Figure 2A) is dominated by intense peaks related to human keratins (see peak signals at _m/z_ 1064.6, 1234.6, 1262.5, 1302.6, 1357.6, 1707.7, 1838.8) that contaminated the artwork's surface. The identity of keratin-type peptides was achieved through RPLC coupled to tandem MS experiments. As an example, the tandem MS spectrum of the doubly charged ion at _m/z_ 854.7 eluting at 12.97 min (Figure S1, Supplementary Material) strongly agrees with the sequence GSIGGGGFSSGGFSGGSFSR of keratin type I cytoskeletal 10, _Homo sapiens_, corresponding to the monocharged ion at _m/z_ 1707.7 detected in the MALDI-MS spectrum (see Figure 2A). Notably, several intense peaks of peptides were generated by trypsin/chymotrypsin auto- and mutual digestion such as _m/z_ 1016.5, 1045.5, 1092.4, 1493.6, and 1541.5 (see Figure 2A), thus suggesting the very low abundance of proteinaceous material in this examined sample and investigated zone. These assignments were supported by Protein Prospector--MS Digest and again verified by RPLC-ESI-CID tandem MS experiments. For example, the ion at _m/z_ 1541.6 (_m/z_ 771.6 as doubly charged ion) corresponds to the peptide IVN(deamidated)GEEAVPGSWPW belonging to the protein chymotrypsinogen A, _Bos taurus_ (Figure S2). Besides interfering signals, both spectra of Figure 2 share several other minor peaks that successfully match with ovalbumin (i.e., _m/z_ 1345.7, 1581.6, 1687.8, 1773.9, 1858.9, and 1859.9), ovomucoid (_m/z_ 1106.5), ovotransferrin (_m/z_ 1179.6), and vitellogenin I and II at _m/z_ 1079.5 and 1179.6, respectively. The search by Protein Prospector--MS Fit corroborated the above results of _G. gallus_ taxonomy. Egg white chymotryptic peptides in the blue layer sample were further confirmed by data-dependent RPLC-ESI-MS/MS analysis. The occurrence of a peak signal at _m/z_ 930.62+, eluted at 14.94 min and corresponding to _m/z_ 1859.8 in the MALDI-MS spectrum, was recognized as the peptide ELIN(deamidated)SWVESQTNGIIR derived from ovalbumin of _G. gallus_ (Figure S3). Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine or glutamine is converted to carboxylic derivative, thus producing a +0.984 Da mass shift. According to literature, deamidation is the main non-enzymatic modification occurring during protein aging along with oxidation, both commonly observed in proteins from ancient artworks [28]. From the very low abundance of the sampled proteins, one could infer that egg white was used as a protective coating rather than as the unique binder, thus the micro-invasive sampling on this point was carried out.