A novel strategy
based on in situ 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). In situ 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.
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