Vlad III the Impaler

Preface
The story of Count Dracula, the most famous feeding on the blood of the living to live forever, is certainly one of Europe’s bloodiest legends, described by the Irish writer Bram Stoker in his book of 1897. (1) Although the nature of this inspiration has been contentious, some scholars and historians theorized that the monstrous legend in Stoker’s famous novel would partly be inspired by a historical figure: The Romanian prince Vlad III, Voivode of Wallachia during the 1400s, also known as Vlad the Impaler, or Vlad Drăculea (the Son of the Dragon). (2) The theory that Vlad III and the Dracula of Stoker’s novel were the same person was developed and popularized by historians Radu Florescu and Raymond T. McNally in their book “In Search of Dracula” (The New York Graphic Society, 1972), but according to different historians and literary scholars, the two Draculas do not really have much in common. On the other hand, the historical figure who shares a name with the literary icon, though there is no real evidence of his alleged vampirism, was no less fearsome. This Prince of Wallachia, in the Carpathian region, reigned with terror over his territory in the 15th century. Vlad Dracula the Impaler (Vlad Tepes in Romanian) was a brutal and sadistic prince-like military leader famous for torturing his foes. Local legends say that he impaled many Ottoman enemies and a lot of people he suspected of plotting against him. By some estimates, he was responsible for the deaths of more than 80,000 people in his lifetime, a large percentage of them by impalement. Centuries later, the sinister reputation of Vlad the Impaler took on a new life when Stoker came across the name Dracula in an old history book, learned that it could also mean “devil” in Walachia, and gave the name to his fictional . However, today, Vlad III is something of a national hero in Romania, where he is remembered for defending his people from foreign invasions, whether Turkish soldiers or German merchants. In light of this preface, it is undeniable that, thanks to the popularization of the image of Bram Stoker’s Dracula, Prince Vlad III himself has also become very famous and immortal, although not in the same way as his literary counterpart.

Introduction
The interest of scientists in analyzing items (paintings, documents, ceramics, fossils, etc.) of World Cultural Heritage has been exponentially increasing since the beginning of the new millennium. Over the past two decades, studies of ancient biomolecules (mainly, but not only, proteins) have opened an important window into the past, improving our understanding, for example, of the evolutionary history, (3) the diet habits of our ancestors, (4) the past human diseases, (5) or the knowledge of the materials used in a painting. (6) These studies have grown considerably in tandem with the development and use of increasingly sophisticated and sensitive technologies and analytical tools. Particularly, the increased number of scientific publications about the characterization of ancient proteins is strictly linked to the impressive technological development, in terms of performance and sensitivity, of mass spectrometry (MS) together with the improvements of protein databases and bioinformatics tools, that has expanded the application of MS-based approaches also to the World Cultural Heritage. Modern mass spectrometers are able to detect and characterize many proteins with extremely high sensitivity even in very complex mixtures, and on very limited amounts of sample, typically less than a few micrograms of crude extract. On the other hand, it is well known that one of the most important issues of the analysis of ancient samples is represented by the contamination of exogenous proteins, mainly constituted by those coming from the post-discovery of the item or from the proteomic analysis laboratory. Therefore, the challenge of discerning contaminant proteins from endogenous ancient molecules becomes of fundamental importance. The deamidation process of asparagine and mainly of glutamine residues (showing a slower deamination rate) is the most applied marker of aging in many archaeological and paleontological studies. (7,8) In addition, the different patterns of degradation and diagenetic chemical modifications (DCMs) affecting ancient proteins, as opposed to potential modern contaminants, are used as authentication and validation criteria for the results. (9,10) Another important challenge is related to the inestimable value of World Cultural Heritage samples, which should be analyzed without damage or contamination. Therefore, the sampling techniques adopted have to be minimally destructive or non-destructive. Indeed, although modern high-performance mass spectrometers require very low amounts of sample, most of the actual protocols of analyses remain invasive, requiring the destruction of part of the ancient items under study; a practice that is (obviously) discouraged by most museums. Consequently, during the last decade, different minimally invasive methods have been developed to harvest and investigate ancient samples. (11−14) A promising non-invasive technique, which at present appears to allow full exploration of any item in the Cultural Heritage arena without any damage or contamination, is known under the acronym EVA (ethylene-vinyl acetate). (15) It consists of a plastic film of ethylene-vinyl acetate studded with strong cation and anion exchangers as well as with C18 and C8 hydrophobic resins which, when applied to any type of surface, is able to extract proteins and small molecules. Then, the harvested molecules can be eluted and characterized via MS. This non-invasive approach coupled with MS-based methods has been applied with promising results to documents, clothing items, and ancient tissue remains. (16−18) Here, we report the results of the MS characterization of the peptides and proteins harvested by the EVA technology applied to three letters of the 15th century that were written to the burghers of Sibiu (Romania) by a man describing himself as “prince of the Transalpine regions” and signed the letters as Vlad Dracula. The characterization of proteins extracted from these three letters provided a window into Wallachia (part of today’s southern Romania) of the second half of the 15th century, a period of military turbulence, also characterized by diseases and epidemics─and not only. The identification of some human peptides and proteins harvested from the letters investigated allowed us to uncover more about Vlad Dracula the Impaler. As described by the papal legate Nicholas of Modrussy, (19) “he was not very tall, but very stocky and strong, with a cruel and terrible appearance, a long straight nose, distended nostrils, a thin and reddish face in which the large wide-open green eyes were framed by bushy black eyebrows, which made them appear threatening.” Two green eyes that, according to some stories, sometimes cried tears of blood, a physical condition, today known as hemolacria, which causes a person to produce tears that are partially composed of blood.