The molecular characterization of samples from works of art can provide valuable insights into the composition of ancient restoration materials and their conservation state. Here, we present a novel analytical protocol for the molecular characterization of a specific adhesive used in historical painting restoration, known as "glue lining pastes." Due to the high molecular complexity of these adhesives, we propose a multistep extraction protocol to recover and fractionate from a single microsample the three main classes of biomolecules contained in glue pastes (lipids, polysaccharides, and proteins). High-performance separation coupled with high-resolution mass spectrometry (MS) techniques were applied to the isolated fractions to identify specific components. The proposed method was optimized using test specimens of various traditional glue pastes applied to canvases and successfully applied to a historical glue paste sample from the 17th-century painting "La fuga in Egitto," part of the Pagliara collection at the University Suor Orsola Benincasa (Naples, Italy). The data collected in this work provide insights into the specific recipe used for adhesive preparation, supporting artistic and historical interpretations and contributing to a broader understanding of old restoration practices. Data are available via ProteomeXchange with the identifier PXD051480.
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