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

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⟩.自新世纪伊始，科学家对世界文化遗产遗存的分析研究兴趣呈指数级增长态势。此类研究的发展与精密灵敏技术的开发应用同步推进，例如高分辨率质谱（high-resolution mass spectrometry, MS），以及缩写为EVA（ethylene-vinyl acetate，乙烯-醋酸乙烯酯）的非侵入无损伤采集技术。本文报道了利用EVA技术采集的肽类与蛋白质的质谱表征结果，该技术被应用于瓦拉几亚大公弗拉德三世（亦被称为穿刺公弗拉德、弗拉德·德古拉）于1457年与1475年撰写的三封书信。通过监测肽类的脱酰胺水平差异及其他成岩化学修饰类型，可有效区分"原始"内源性肽与污染物肽。对从这些书信中提取的古代蛋白质进行表征，使我们得以探究15世纪下半叶瓦拉几亚地区的环境状况——该区域曾被视为士兵、移民与旅行者的交汇之地，这些人群不仅可能携带贸易商品与文化传统，还可能传播疾病与流行病。此外，从这些书信中鉴定出的大量人类肽类与蛋白质，使我们得以进一步了解穿刺公弗拉德·德古拉。具体而言，实验数据表明他可能曾罹患呼吸道及/或皮肤的炎症性疾病。此外，尽管蛋白质组学数据尚不全面，但结合相关史料记载，其结果提示他或许还曾罹患一种名为血泪症（hemolacria）的病理状态，即可能分泌混有血液的眼泪。值得注意的是，不可否认中世纪时期曾有多人触碰过这些书信，但可以合理推测，其中丰度最高的古代蛋白质应与撰写并签署这些书信的穿刺公弗拉德大公相关。本研究数据集已提交至ProteomeXchange数据库，编号为⟨PXD041350⟩。