Fate of egg white proteins in aged painting layers by UV-induced processes: a comprehensive proteomic study

Understanding the aging of pictorial binders is essential for preserving artworks and identifying the chemical changes that affect their stability. Among historical binders, egg tempera was widely used due to its durability, yet protein degradation over time weakens the painting layer, especially when interacting with inorganic pigments. This study simulated the natural aging of egg-based paintings by exposing non-pigmented egg white and egg white mixed with calcium carbonate (white pigment) or hematite (red pigment) to UV-Vis irradiation and 50 °C for up to 864 h. ATR-FTIR spectroscopy revealed structural protein changes, with Amide I and II spectral shifts indicating increased intermolecular β-sheet formation, a hallmark of protein aggregation. Proteomic analysis performed using three different enzymes (Trypsin, AspN, and GluC) reinforced these findings. MALDI-ToF-MS and LC-MS/MS revealed a marked decline in the signal-to-noise ratios of specific peptides, highlighting the increasing difficulty of extracting and digesting aggregated proteins. This challenge underscores the extent of structural protein alterations over time. Furthermore, the study identified aspecific and semi-specific peptides, predominantly originating from the most aging-susceptible regions of ovalbumin, confirming that protein degradation occurs alongside aggregation. Notably, prolonged aging conditions induced previously unreported non-enzymatic post-translational modifications (PTMs) in aged proteinaceous painting binders, such as methylation, formylation, and diglycyl-modifications. These modifications likely contribute to the destabilization of the pictorial layer, shedding new light on the intricate chemical transformations that occur in aged egg tempera paintings.