Count Dracula Resurrected: Proteomic Analysis of Vlad III the Impaler’s Documents by EVA Technology and Mass Spectrometry

[Image: see text] The interest of scientists in analyzing items of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. These studies have grown considerably in tandem with the development and use of sophisticated and sensitive technologies such as high-resolution mass spectrometry (MS) and the non-invasive and non-damaging technique, known under the acronym EVA (ethylene-vinyl acetate). Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters written in 1457 and 1475 by the voivode of Wallachia, Vlad III, also known as Vlad the Impaler, or Vlad Dracula. The discrimination of the “original” endogenous peptides from contaminant ones was obtained by monitoring their different levels of deamidation and of other diagenetic chemical modifications. The characterization of the ancient proteins extracted from these documents allowed us to explore the environmental conditions, in the second half of the 15th century, of the Wallachia, a region considered as a meeting point for soldiers, migrants, and travelers that probably carried not only trade goods and cultural traditions but also diseases and epidemics. In addition, the identification of many human peptides and proteins harvested from the letters allowed us to uncover more about Vlad Dracula the Impaler. Particularly, the experimental data show that he probably suffered from inflammatory processes of the respiratory tract and/or of the skin. In addition, proteomics data, although not exhaustive, suggest that, according to some stories, he might also have suffered from a pathological condition called hemolacria, that is, he could shed tears admixed with blood. It is worth noting that more medieval people may have touched these documents, which cannot be denied, but it is also presumable that the most prominent ancient proteins should be related to Prince Vlad the Impaler, who wrote and signed these letters. The data have been deposited to the ProteomeXchange with the identifier ⟨PXD041350⟩.