Hydrogel Extraction with Dual-Enzyme Digestion of Proteinaceous Binders: the Key for Reliable Mass Spectrometry Investigations of Artworks.

A novel strategy based on dual-enzyme digestion of paint layer proteinaceous binders is introduced for faster and more confident identification, resulting in a bottom-up proteomics approach by MALDI-TOF mass spectrometry (MS). sampling/extraction of proteinaceous binders using small pieces of a hydrophilic gel, previously loaded with trypsin and chymotrypsin proteolytic enzymes, was successfully exploited. Along with minimal invasiveness, the synergy of both enzymes was very useful to increase the number of annotated peptide peaks with their corresponding amino acid sequence by database search and subsequent MALDI-TOF/TOF analysis. The protocol was initially aimed at enhancing the identification of egg-based binders and then validated on fresh and aged model pictorial layers; an increased protein coverage was significantly attained regardless of the used painting binders. Optical microscope images and spectrophotocolorimetry analysis evidenced that the painting layers were not damaged or altered because of contact/sampling without leaving hydrogel residues. The proposed protocol was successfully applied on a painted altarpiece "Assumption of the Virgin" dated to the XVI century and on an angel statue of the Nativity crib dated to the XII century, both from Altamura's Cathedral (Apulia, Italy). The occurrence of various protein binders of animal origin was easily and reliably ascertained.