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Proteomics reveals proteins in paint Art historians, archeologists, and museum curators need to know the biological compositions of historical works for the sake of proper documentation and preservation. However, current analytical methods give little quantitative or structural information about the biomolecules present in old paintings or ancient pieces of pottery. In this issue of Analytical Chemistry (pp 1494– 1502), Cécile Cren-Olivé and colleagues at the University of Science and Technology of Lille (France) and the Centre de recherche et de restauration des musées de France describe a method that takes advantage of proteomics to identify proteins in paints from the Renaissance era. Paints consist of pigments and binders. Pigments tend to be inorganic materials like minerals, but the binders can be organic compounds, such as milk, eggs, or animal-based glue. Analytical methods exist to pinpoint the presence of proteins in paints, but they don’t allow the unambiguous identification of the proteins. For example, histochemical and staining approaches only highlight the presence of proteins. Chromatographic techniques give the aminoacid composition, but the amino-acid composition doesn’t help to identify the proteins. Cren-Olivé says spectroscopic approaches, such as IR and Raman, are “very difficult to use in the case of egg [binders] because some of the components, like egg yolk, have exactly the same triglyceride profile as linseed oil.” The work described in the current Analytical Chemistry paper began in the early 1980s when one of the authors, Elizabeth Martin, was asked by the curators of the Louvre museum in Paris to accurately identify proteins in flakes of paint from Renaissance paintings. But given the analytical methods at hand, the investigators weren’t able to precisely determine the kinds of proteins present in the paint flakes. Cren-Olivé says,
“With the appearance of proteomics, now we can answer this question.” Cren-Olivé and colleagues first created formulations of paints to use for the development of their protein-extraction
A new protein-extraction method determined that Benedetto Bonfigli’s triptych The Virgin and Child, St. John the Baptist, St. Sebastian contained both egg whites and yolks as the paint binders.
method. They used lead white as the pigment and either whole egg or a mixture of ovalbumin and linseed oil as binders. From their analyses, they found the most efficient way to extract the binder proteins was to use a mortar and pestle to grind the paint sample into a fine powder with a synthetic, commercial resin. The powder was suspended in a 1% trifluoroacetic acid solution and incubated several times in an ultrasonic bath. The investigators first studied the protein extraction efficiency by MALDI TOFMS. To ensure that they could unambiguously identify the extracted proteins, Cren-Olivé and colleagues then digested the extracted proteins with trypsin and analyzed the peptides by LC/MS/MS. Upon confirmation that their technique worked, the investigators turned their attention to ~10-µg paint samples from 2 paintings—Benedetto Bonfigli’s
15th-century triptych The Virgin and Child, St. John the Baptist, St. Sebastian and Niccolo di Pietro Gerini’s 14thcentury painting The Virgin and Child. By using their extraction and LC/MS/ MS methods, Cren-Olivé and colleagues demonstrated that whole eggs were the binding agents in the two samples from the Renaissance paintings. The researchers were careful to specify that the Bonfigli paint sample came from St. Sebastian’s left elbow and that the Gerini sample came from the Virgin’s dress. The precise location of the paint samples had to be accurately cataloged because, as Cren-Olivé explains, “We have to take different flakes from the same painting to evaluate if the painter had always used the same binder for all the colors or if he chose to modify the binder to obtain a different chromatic effect.” Cren-Olivé and colleagues are now using their analytical methods to study remnants of pottery from archeological digs. They are particularly interested in studying the diet of ancient humans. For instance, they are analyzing milk proteins in shards of pottery to determine whether the milk came from cows, goats, or sheep. The source of the milk could shed light on the types of domesticated animals ancient humans kept in the region where the pottery pieces were found. In a related study, the investigators are looking at traces of lipids in bits of pottery dating from Neolithic times by using their proteomics method for the analyses. They would like to know for how long certain types of human diets existed. “From the south of Europe, [such as] Italy and south of France, we were able to identify lots of lipids from vegetable oils, like olive oil. From the north of France, in two out of three [pottery] samples, we identified animal fats,” explains Cren-Olivé. “It seems to show that even at this [Neolithic] time, the dietary habits that are currently [established] in Europe were already present.” a —Rajendrani Mukhopadhyay
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