Ahead of display, a non‐original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post‐translational modifications detected several photo‐oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR‐FTIR in a cross‐section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.

Dyes derived from brazilwood (Caesalpinia spp.) are known to have been used in a diverse range of objects, from Medieval European textiles to North American First Nations objects, while pigments made from brazilwood feature in the palette of a number of painters, including Rembrandt and Van Gogh. For almost two decades, an unknown marker has been used to detect brazilwood colourants in historical objects. Limited sampling opportunities mean that the identification of the chemical structure of this marker has eluded scientists to date. Using a combination of synthesis, UPLC-ESI-MS/MS, HPLC, NMR and GC-MS, the identity of this unknown marker was confirmed as the benzochromenone, urolithin C. Structural identification provides a reliable reference compound for use across a range of analytical techniques employed in the cultural heritage sector and will enable the future development of non-destructive techniques for its identification on high-status objects.

The fugitive nature of the colorants obtained from sappanwood (Caesalpinia sappan L.) or the South American species commonly known as ‘brazilwoods’ (including other Caesalpinia species and Paubrasilia echinata (Lam.)) makes the identification of brazilwood dyes and pigments in historic artefacts analytically challenging. This difficulty has been somewhat alleviated recently by the recognition and structural elucidation of a relatively stable marker component found in certain brazilwood dyes and pigments—the benzochromenone metabolite urolithin C. This new understanding creates an ideal opportunity to explore the possibilities for urolithin C’s localization and identification in historical artefacts using a variety of analytical approaches. Specifically, in this work, micro-destructive surface-enhanced Raman spectroscopic methods following a one-sample two-step (direct application of the colloid and then subsequent exposure of the same sample to HF before reapplication of the colloid) approach are utilized for the examination of four historical brazilwood dyed textiles with the results confirmed via HPLC-DAD analysis. It is shown that characterization of reference urolithin C is possible, and diagnostic features of this molecule can also be traced in faded historical linen, silk and wool textiles, even in the presence of minor quantities of flavonoid, indigoid and tannin components. The exploitation of the same micro-sample through a series of SERS analyses affords a fuller potential for confirming the characterization of this species.

Ahead of display, a non‐original layer was observed on the surface of a fragment of a wall painting by Ambrogio Lorenzetti (active 1319, died 1348/9). FTIR analysis suggested proteinaceous content. Mass spectrometry was used to better characterise this layer and revealed two protein components: sheep and cow glue and chicken and duck egg white. Analysis of post‐translational modifications detected several photo‐oxidation products, which suggest that the egg experienced prolonged exposure to UV light and was likely applied long before the glue layer. Additionally, glycation products detected may indicate naturally occurring glycoprotein degradation or reaction with a carbohydrate material such as starch, identified by ATR‐FTIR in a cross‐section of a sample taken from the painting. Palaeoproteomics is shown to provide detailed characterisation of organic layers associated with mural paintings and therefore aids reconstruction of the conservation history of these objects.