Pyrolysis Compound-Specific Isotope Analysis of Proteins in Aged Paintings: Diketopiperazine δ13C Values as Collagen...

Proteins have been used as adhesives in art throughout history, but determining their source in aged paintings is challenging. Moreover, the distinction between terrestrial and aquatic animal glues is only achievable with proteomic approaches. Compound-specific isotope analysis (CSIA) of collagen, used in the neighboring field of archeology for analogous purposes, has so far required too much sample to be used for painted objects. However, this paper presents a methodology that enables a reduction of sample size by about 3 orders of magnitude (from mg to μg), widening the application of CSIA to rare and fragile objects in our cultural patrimony. Based on direct inlet pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (DIP-GC-C-IRMS), the method targets the δ13C values of pyrolytic protein markers, especially diketopiperazines (DKPs), reported here for the first time. The isotopic imprint of modern references for rabbit skin and isinglass glues, casein, and chicken and duck eggs was confirmed through established elemental analysis (EA)-IRMS and GC-C-IRMS methods. As expected, bulk and individual amino acid (AA) δ13C and δ15N values for terrestrial animal glues exhibited a higher depletion than those for marine glues, a relationship retained by the DKP δ13C values. Furthermore, the ground layers of two 16th-century Italian paintings from the National Gallery collection yielded δ13CDKP values consistent with terrestrial animal glue, confirming that the isotopic signature of these compounds is retained during aging and degradation.