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Historical stringed musical instruments such as violins, violas, and violoncellos are a unique class of cultural heritage objects. The production of these precious musical instruments reached its peak of prosperity in the 16–18th century in a northern Italy city, Cremona, represented by the great Masters of violin-making art. The multi-layered coating system and the pre-treatments applied to the surfaces of wood substrates are assumed to be related to the special sounds and esthetics of these antique instruments. However, due to the absence of written historical records, their construction and finishing methods are still a mystery. For this purpose, chemical analyses are useful to recover historical recipes by providing information about the material composition, methods of manufacture, and past restoration and maintenance procedures. Pyrolysis gas chromatography-mass spectrometry provided useful information on material composition by using specific markers, but protein determination remains an open analytical problem. A comprehensive study on various proteinaceous materials was undertaken to evaluate the performance of off-line pyrolysis combined with solid-phase microextraction with and without on-fiber silylation. The molecular markers were selected for each method. The methods were applied to historic instrument samples from Jacob Steiner, Luigi Baioni, Andrea Guarneri, Francesco Ruggeri, Lorenzo Storioni instruments and a violin from Pietà of Venice. For the first time, the presence of proteinaceous materials was found by analytical pyrolysis in Stainer, Guarneri, Ruggeri, Storioni and probably Pietà of Venice. Different combinations of the markers were observed in each specimen, with pyrocoll, diketopiperazines or silylated 3-hydroxypyridine the most common. Besides collagen, markers indicative of wood components (methoxy phenols, anhydrosugars), gums (anhydrosugars), and resins (dehydroabietic acid, larixyl acetate) provided a complete picture of the organic materials in stringed instruments obtainable by a single analysis of a micro-sample. [Display omitted] • Proteinaceous materials in historical bowed string instruments revealed by pyrolysis silylation. • Silylated pyrolysis markers for collagen in historical bowed string instruments. • Composition of organic materials in historic musical instruments revealed by pyrolysis silylation. • Silylated anhydrosugars indicative of the gum presence in the Storioni small violin. • GC-MS data were provided for pyrolysis products indicative of proteinaceous materials. [ABSTRACT FROM AUTHOR]