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We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of a 1200-1400 AD Inupiat potsherd fragment from Point Barrow, Alaska, by a dedicated proteomics approach. The four main steps of a proteomics analysis, (i) protein extraction from biological samples, (ii) protein hydrolysis using a hydrolase enzyme, (iii) nanoLC, nanoESI MS, and MS/MS analysis of the generated peptides, and (iv) protein identification using protein databank proceeded from genomic data, have been optimized for archeological remains. Briefly our procedure starts by grinding the potsherds, extraction with 1% trifluoro-acetic acid, digestion with excess of trypsin, nanoLC, nanoESI Fr-ICR analysis, and data mining by homology search. The developed conditions were evaluated on protein extracts from remains obtained by heated muscle tissues and blubbers of different seal and whale species, these samples representing the main diet sources of the Eskimo population. Most of the proteins were identified by sequence homology to other species due to the lack of cetacean and pinniped proteins in the databanks. More interestingly, two proteins, myoglobin and hemoglobin, respectively, identified in muscle tissue samples and blubber samples highlight several specific peptides of cetacean and pinniped species; these peptides are significant to prove the presence of these marine species in the analyzed samples. Based on the developed methodology and on protein identification results obtained from the heated seal/whale muscle tissues and blubbers, the analysis of the clay matrix of a 1200-1400 AD Inupiat potsherd fragment from Point Barrow was investigated. The described method succeeds in identifying four peptides corresponding to the harbor seal myoglobin (species Phoca vitulina) with a measured mass accuracy better than 1 ppm (MS and MS/MS experiments) including one specific peptide of the cetacean and pinniped species and one specific peptide of the seal species. These results highlight, for the first time, a methodology able to identify proteins from a few milligrams of archeological potsherd buried for years; the obtained results confirm the presence of a seal muscle tissue protein in this Punuk potsherd.

A new multistep analytical methodology is described in this paper for the precise identification of triacylglycerols, which are biomarkers of dairy products and subcutaneous fats, that may be chemically identified in archaeological pottery. It consists of the analysis of the total lipid extract from different kinds of fats by high-temperature gas chromatography, performed in order to select the ceramic vessels in which animal fats are well preserved, followed by nanoelectrospray QqTOF mass spectrometry that allows for distinguishing the specific origins of the lipids detected (namely, cow, sheep, or goat). The analysis of model samples, cow and goat dairy products and cow and sheep adipose fats, was successfully achieved. The fatty acid composition of each triacylglycerol was identified, which allowed for the discrimination of subcutaneous fats and dairy fats and distinguishing between cow and goat milk. This methodology was then applied to archaeological samples, and the presence of goat milk, cow milk, and possibly sheep subcutaneous fat was assessed based on the discriminating criteria found on modern fats.

The presented work proposes a new methodology based on proteomics techniques to identify proteins in old art paintings. The main challenging tasks of this work were (i) to find appropriate conditions for extracting proteins from the binding media without protein hydrolysis in amino acids and (ii) to develop analytical methods adapted to the small sample quantity available. Starting from microsamples of painting models (ovalbumin-based, which is the major egg white protein, and egg-based paintings), multiple extraction solutions (HCI, HCOOH, N[H.sub.3], NaOH) and conditions (ultrasonic bath, mortar and pestle, grinding resin) were evaluated. The best results were obtained using a commercial kit including a synthetic resin, mortar and pestle to grind the sample in an aqueous solution acidified with trifluoroacetic acid at 1% with additional multiple steps of ultrasonic baths. The resulting supernatant was analyzed by MALDI-TOF in linear mode to verify the efficiency of the extraction solution. An enzymatic hydrolysis step was also performed for protein identification; the peptide mixture was analyzed by nanoLC/ nanoESI/Q-q-TOF MS/MS with an adapted chromatographic run for the low sample quantity. Finally, the developed methodology was successfully applied to Renaissance art painting microsamples of ~10 [micro]g from Benedetto Bonfigli's triptych, The Virgin and Child, St. John the Baptist, St. Sebastian (XVth century), and Niccolo di Pietro Gerini's painting, The Virgin and Child (XIVth century), identifying, for the first time and without ambiguity, the presence of whole egg proteins (egg yolk and egg white) in a painting binder.

An investigation is performed on the redox properties of a series of hydroxychalcones, a group of polyphenols abundantly present in plants, by cyclic voltamemetry. Oxidation potentials of the phenolates correspond to species that are very easy to oxidize, lead to the rapid formation of nonradical species, underlining the potential antioxidant properties of these molecules

To better detect and identify beeswax in ancient organic residues from archaeological remains, we developed a new analytical methodology consisting of the analysis of (i) the trimethylsilylated organic extract by GC/MS and (ii) the crude extract by ESI-MS. Selective scanning modes, such as SIM or MRM, permit separate quantification of each chemical family (fatty acids, monoesters, monohydroxyesters, and diesters) and allow an improvement in sensitivity and selectivity, allowing the crude extract to be treated without further purification. GC/MS (SIM) was revealed to be a powerful method for the detection of components, with a detection limit down to a total lipid extract in the range of ~50 ng in a complex matrix, such as archaeological degraded material, whereas ESI-MS/MS is instead used for the detection of nonvolatile biomarkers. Identification by GC/MS (SIM) and ESI-MS/ MS (MRM) of more than 50 biomarkers of beeswax in an Etruscan cup at the parts-per-million level provides the first evidence for the use of this material by the Etruscans as fuel or as a waterproof coating for ceramics.

Adhesives made from natural substances such as resins, tars, and waxes are found during excavations on archaeological sites dating back to prehistoric periods. Until now, their analysis was mainly performed by gas chromatography, possibly coupled to mass spectrometry, after extraction, purification, and derivatization of the samples. To minimize sampling and sample preparation of ancient organic remains, which are often preserved in tiny amounts, we have directly analyzed archaeological samples from Bronze and Iron Age periods by direct inlet electron ionization mass spectrometry. A series of contemporary natural and synthetic substances, including pine and pistacia resins, birch bark tar, beeswax, and plant oils, possibly used for adhesive fabrication during ancient times, was also investigated with the same technique as reference materials. Despite the complexity of their chemical composition, pine resin and birch bark tar were clearly identified in archaeological samples. Furthermore, mass spectrometry has been shown to be efficient for the identification of glues made of a mixture of beeswax, presenting a series of mass spectral peaks assigned to long-chain esters, and birch bark tar, whose mass spectrum presents characteristic peaks of lupane compounds. The intentional mixing of birch bark tar and beeswax during prehistory is reported here for the first time.

The chemical reactivity of an electroactive neutral compound in the conditions of electrospray mass spectrometry was studied by carrying out preparative experiments. The residue obtained by spraying a micromolar methanolic solution of 9-nitroanthracene onto a metallic plate in negative mode (cathodic capillary) was collected and then analyzed by GC/MS, showing that both reduction and oxidation products were formed. To distinguish between the two processes, the two functions of the metallic plate, definition of the electric potential and storage of products, were separated by using a copper-bronze grid as counter electrode and a silica TLC plate, located a few millimeters behind, as collector. The collected products were thus stored away from the counter electrode, avoiding their possible reoxidation at this site. The amount of anthracene, a reduction product of 9-nitroanthracene, which was detected only in trace amounts with the metallic plate collector, increased significantly with the new device. Thus, anthracene can be viewed as the result of a capillary process whereas 9,10-anthraquinone can be estimated as mainly due to further oxidation at the collector. Furthermore, in deuterated solvents no incorporation was detected, suggesting that some reaction steps take place at the collector.

The negative-ion mass spectra of arylazo sulfides ArN=NSR have been investigated. The results showed that their radical anions (ArN=NSR.-) generate the radical anions of the corresponding sulfides (ArSR.-) and/or fragments therefrom. The reaction pathway has been proposed to occur via an electrostatically thiolate ion-aryldiazenyl radical complex, in competition with dissociation of the complex into separated species.

Ketone resins such as cyclohexanones and methylcyclohexanones that are found in art paintings can be characterized using pyrolysis/gas chromatography/mass spectrometry. This technique detects resin products even at exceedingly small amounts of test samples. Furthermore, examination of the pyrolysis products and the resin compounds shows that 1, 5-diketone patterns, 3-hydroxy, 5-hydroxy and 5,6-unsaturated carbonyl groups undergo pyrolysis through a cyclic six-centered mechanism which leads to the abstraction of an alpha proton in the ketone functions.

The physical chemistry and the free radical chemistry of flavan-3-ols are extensively investigated by laser flash photolysis and cyclic voltammetry studies. The results are very useful in discussing biological and biochemical phenomenon and give a new insight into the antioxidative properties of flavan-3-ols.