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The presented work proposes a new methodology based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extracting proteins from the binding media without protein hydrolysis in amino acids and (ii) to develop analytical methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extraction solutions (HCl, HCOOH, NH₃, NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a commercial kit including a synthetic resin, mortar and pestle to grind the sample in an aqueous solution acidified with trifluoroacetic acid at 1% with additional multiple steps of ultrasonic baths. The resulting supernatant was analyzed by …