Determination of animal species origin of blood in artworks by LC-MS/MS

This study introduces an innovative approach to the long-standing challenge of determining the animal origin of blood used in artworks - an issue of central relevance to art historians aiming to understand historical techniques, symbolic meanings, and the cultural traditions associated with artistic materials. Using LC-MS/MS analysis, species-specific peptide sequences of blood proteins were identified, allowing for the discrimination of seven animal species (cat, cow, dog, goose, hen, human, and pig). This analytical approach was successfully applied to a series of model samples containing blood, confirming both the proteinaceous composition and the animal origin of the blood. Furthermore, the method enabled the identification of pig blood in historical samples taken from Japanese and Chinese lacquer artefacts dating from the 18th and 19th centuries. The novelty of this work lies in a new strategy for data evaluation that facilitates the creation of custom peptide databases tailored to distinguish specific animal species. This approach overcomes a major limitation in proteomic studies - namely, the lack of complete sequence data for many animals - by incorporating homologous sequences from closely related species. The strategy demonstrates a high degree of effectiveness when implemented within a clearly defined group of animal species, such as those historically utilised in the production of blood-based artistic materials. This approach offers a novel pathway for the molecular identification of animal origin in cultural heritage contexts and establishes a robust foundation for future interdisciplinary investigations bridging art history, conservation science, and molecular biology.