When historians study texts, whether they’re ancient records or more recent manuscripts, they look to the words on the page to tell them about the life and times of the authors. But words aren’t all that writers leave behind. Metabolites from the medications they were taking, bacterial proteins from microorganisms that may have been in their surroundings, and even traces of their meals accumulate on the pages of these old documents—and can sometimes tell a different story.
An international team of scientists has been using a novel, nondestructive analytical technique to suss out such proteins and small molecules and uncover their biochemical secrets. The researchers’ most recent molecular sleuthing has given them insight into the outbreak of plague that struck Milan in 1630 and brought them to the deathbed of Russian writer Anton Chekhov.
The analytical technique uses small, thin plastic diskettes, known as an EVA films because they’re made from ethyl-vinyl acetate. The scientists moisten a diskette with distilled water and place it on the surface of an object for study, such as a painting, manuscript, or article of clothing. Beads of anion and cation exchange resin embedded within the EVA film pick up proteins and other charged molecules from the object’s surface. Similarly, hydrophobic C8 resins in the EVA films gather oily molecules from the surface. The researchers then remove the film from the object, wash the collected compounds from its surface, and analyze them with liquid chromatography and mass spectrometry (Anal. Chem. 2017, DOI: 10.1021/acs.analchem.6b03722).
In a report published last year, the team showed the technique can be used on paints on canvases, frescoes, and wood; bone; parchment; and linen without damaging these objects or leaving behind any residue.
“We developed this method so that we wouldn’t produce any damage on the surface of these cultural heritage documents,” says Pier Giorgio Righetti, a chemist at Polytechnic University of Milan, who leads the EVA film research. “We feel that this is perhaps the only safe way to explore the surface of many documents belonging to human history.”
Using this technique, one can learn what health conditions may have contributed to an author’s masterpiece, says Gleb Zilberstein, a scientist at the sensors firm Spectrophon who works with Righetti. For example, Zilberstein points to work the researchers published in 2015 in which they found traces of morphine, morphine metabolites, and protein biomarkers of kidney disease on the original manuscript of Mikhail Bulgakov’s “The Master and Margarita,” a famous Soviet satire (J. Proteomics 2015, DOI: 10.1016/j.jprot.2015.11.002).
Bulgakov finished the book just four weeks before his death in 1940. Righetti and Zilberstein’s team showed that the author was able to ease the pain of his chronic kidney disease and finish his novel with the help of morphine.
Righetti notes that the analytical technique has evolved since that 2015 report. In that paper, the researchers simply placed resin beads directly onto Bulgakov’s manuscript. After the work was published, a scientist wrote to the researchers to point out that they couldn’t be certain they hadn’t left any beads behind on the documents. “He had a point,” Righetti says. “We couldn’t guarantee that some of the beads were not trapped in the threads of the paper.” So they decided to immobilize the beads in the EVA film.
Once the EVA films were ready, Righetti knew exactly what he wanted to study—the outbreak of plague that struck Milan in 1630. “In Italy, everybody knows about the story of the plague in Milan,” he says, thanks to Alessandro Manzoni’s 1827 novel “The Betrothed,” which describes the plague’s grip on the city. The novel, Righetti says, became mandatory reading in Italian schools in the 1870s and has remained so ever since.
In 1630, the plague swept through Italy’s Lombardy region, where Milan is the capital, killing up to 2,000 people each day during its peak from June through September. About 120,000 people—roughly half the area’s population—died during this time. Righetti’s team decided to use the EVA films to study substances left in the margins of death registries kept by humble scribes during the outbreak. Stored in Milan’s State Archives, these books record the names and cause of death of each person that died.
What the researchers discovered surprised them. “These mixtures are very complex,” says Alfonsina D’Amato, the team’s proteomics expert, who works at the University of Milan. She found not only proteins from humans but also proteins from mice and proteins from bacteria other than Yersinia pestis, the species that causes plague (J. Proteomics 2018, DOI: 10.1016/j.jprot.2017.11.028).
Naturally, the researchers expected to find traces of Y. pestis. “In the death registries, about 95% of the people are diagnosed with plague,” Righetti says. But proteomic analysis showed that proteins from the anthrax bacterium were also present on the pages, explaining entries in the registries where people died of high fever.
The team also found proteins from corn, rice, carrots, and chickpeas. Righetti thinks this was the primary food of the poor scribes, who left traces of their meals behind as they worked to frantically record all the deaths around them.
Finally, the researchers found keratin proteins from mice. “We expected to find human keratins,” Righetti says, “but it was totally unexpected to find an almost equal number of keratins from mice.” The most likely explanation, he says, is that at night these rodents would eat table scraps left by the scribes, scurrying over the registry books left open on the table. Once completed, the registries were stored tightly in stacks, so it seems unlikely the mice would have been able to access the pages at any other time, he says.
The team also recently had the opportunity to use EVA films to analyze a blood spot on the shirt Anton Chekhov wore on the day he died, along with letters and a postcard the Russian writer and medical doctor wrote in his final months. The shirt had been safely stored away from environmental contaminants following Chekhov’s death in 1904. The blood spot also contained some dried saliva, in which the researchers were able to detect proteins from Mycobacterium tuberculosis (Proteomics 2018, DOI: 10.1002/pmic.201700447).
Historians have assumed that Chekhov died of tuberculosis, and the researchers’ finding confirms this diagnosis. The team also wondered if he had taken any medications for his ailments but were unable to detect any traces of laudanum—a popular opium tincture used at the time. Righetti says they hope to reexamine the artifacts with an EVA film embedded with beads that might be better at picking up the alkaloids found in opium.
Matthew Collins, who studies proteins in ancient artifacts at the University of Copenhagen and the University of York, says the findings in both reports are intriguing and merit further study. He thinks the EVA films represent a novel approach to analyzing artifacts, but suspects the use of water to moisten the films would give many conservators pause. For example, old parchment, which is made from animal collagen, can turn to gelatin when wet, he says. “It doesn’t happen very often, but when it does happen, it’s positively terrifying.”
Righetti and Zilberstein hope EVA films will catch on for analyzing samples beyond historical artifacts. They think the diskettes could be useful as a crime-scene analysis tool for law enforcement, for example. But they also have other historical objects they’re hoping to study in the future, including manuscripts from 19th-century Russian writer Alexander Pushkin.