Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).

Follow-up on the characterization of peptidic markers in hair and fur for the identification of common North American species.

RATIONALE: Species identification of hair is routinely done by microscopic analysis. Following previous studies that used protein analysis to characterize species markers in hair and wool, the present work aims at covering a larger number of species and to ultimately offer a method for rapid hair identification in forensics and archaeology. METHODS: Hair is mostly made of alpha-keratins; these proteins have only been sequenced in a handful of species and most animal families are under-represented. Using a methodology developed for the characterization of peptidic markers in tissues such as bone (peptide mass fingerprinting or PMF) and commonly applied on collagen, hair from common North American fur-bearing species was analyzed by MALDI-TOF-MS to obtain peptidic profiles. RESULTS: Alpha-keratin peptides that are typically dominant on peptide mass profiles of hair were chosen as markers. Matching peaks were identified for each species tested and compared to known sequences from related organisms whenever possible. The markers were used to create a flowchart to narrow down identification to the family level. CONCLUSIONS: The methodology was developed on a limited numbers of markers chosen for their variability and reliability on the peptide mass fingerprint. In the absence of genetic sequences, this strategy is a quick way to compare species from a common geographic origin. The work presented here was focused on North American species but could be applied to other animal families.

Modeling deamidation in sheep α-keratin peptides and application to archeological wool textiles.

Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wool's α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.

Palaeoproteomics and microanalysis reveal techniques of production of animal-based metal threads in medieval textiles.

Animal-based metal threads were largely used between the 10th and the fifteenth century, in European, Middle Eastern and Far Eastern textile productions for the decoration of textiles and cloths. They belong to a larger group of metal threads, used either as flat threads or wrapped around a fiber core, that were backed by an organic support (animal or paper). This study focuses on the medieval production of metal threads backed by an animal membrane (e.g. gut membrane), or skin. A total of 91 samples were collected from a corpus of 66 textile fragments belonging to 54 catalogued objects. The relevance and novelty of the present study is represented by the combination of proteomics, cross-section analysis, and scanning electron microscopy (SEM-EDS and SEM-µXRF). The diversity of materials and manufacturing techniques found within each typology of thread, respectively, membrane-based metal threads and skin-based metal threads, hinted at different production technologies. Membrane-based threads were found to be invariably made from cattle gut membrane, coated with gilt-silver leaves. A possible sheep glue adhesive was found in a few samples. Skin-based threads were made from either goat or sheep leather, coated with metal leaves or powder. Within the three different types of coatings identified (silver, gold and gilt-silver), gold coatings were the most represented. Goat leather threads were associated with an egg-white binder, while sturgeon glue was identified as adhesive in all sheep leather threads. Collagen glue from other species (cattle, sheep, horse) was occasionally found in mixed adhesives. In two textiles, the finding of human proteins indicates past contamination due to handling or use. The analytical results show coherence between the fabrication patterns of animal-based metal threads and their probable geographical areas of manufacture, indicating that the study of materials and techniques provide further criteria to classify and group textiles, and trace correlations between manufacturing centers within Eurasian territories.

Characterisation of novel α-keratin peptide markers for species identification in keratinous tissues using mass spectrometry.

RATIONALE: In ancient and/or damaged artefacts containing keratinous materials, the species of origin of the materials can be difficult to identify through visual examination; therefore, a minimally destructive methodology for species identification is required. While hair fibres from some species have seen substantial characterisation, others such as horn or baleen have received little or no attention, or lack protein sequences allowing formal identification using proteomics techniques. METHODS: We used the PMF method (Peptide Mass Fingerprinting with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS)) to catalogue and identify diagnostic peptide markers up to the genus level. Sequences were checked using nanoflow liquid chromatography/electrospray ionisation tandem mass spectrometry (nanoLC/ESI-MS/MS) and unidentified peptides were searched against a theoretical database generated by substituting amino acids in keratin sequences. RESULTS: Specific peptides were identified by m/z and sequences characterised whenever possible for a range of species belonging to Bovidae and Camelidae, and for tissues such as baleen and horn. The theoretical database allowed an increase in the number of peptides of up to 10% in species with little genetic information. CONCLUSIONS: A proteomics approach can successfully identify specific markers for the identification of materials to the genus level, and should be considered when identification by other means is not possible. Identification by PMF is fast, reliable and inexpensive.

A simplified sample preparation for hair and skin proteins towards the application of archaeological fur and leather.

Conventional protocols for proteomics analysis usually start by extracting or solubilizing the proteins from their substrates. This step can be challenging for archaeological proteins, when they are heavily contaminated or decayed. The remains of animal fur/leather objects from an early medieval burial in Trossingen (580 CE) from Southwest Germany were submitted to proteomics analysis for species identification. One leather sample (TS3) yielded enough proteins to be identified as cow using a urea-based extraction (method "U"), confirming the microscopic identification. But two other samples (TS1 and TS2), compacted in a greyish brittle matrix with embedded hair visible only under microscope, could not be characterized with that method. A series of tests was performed using reduction/alkylation with tris(2-carboxyethyl)phosphine/chloroacetamide at 95 °C directly on the matrix (method "95C"), with or without the use of paramagnetic beads as cleaning procedure (from the single-pot solid-phase-enhanced sample preparation or SP3). Hair keratins were best recovered in the fur samples when digestion was performed directly on the insoluble fraction after reduction/alkylation. For both samples TS1 and TS2, an ovicaprine species was identified, with TS1 firmly identified as sheep due to the exceptional preservation of keratins and keratin-associated proteins. The simplified protocol also showed improvements on the identification of collagen in the leather sample TS3. SIGNIFICANCE: North European burials had a strong tradition of bodies wrapped or covered in animal skins; textiles, furs, items of leather and other organic materials were essential parts of grave furnishings (as part of the deceased's clothing as well as grave goods) but are mostly only preserved as residues, uncharacterized layers or stains. Even well preserved finds like the waterlogged organic remains from Trossingen show strong limitations for visual identification. Because the traditional protocol was unable to extract proteins efficiently from the soil matrix in which the samples were embedded, a new method was devised that enabled the determination of the sampled fur remains as sheep and the leather fragments as cow leather. Analyses showed that the key step for accessing the proteins in the soiled archaeological samples was heating for 10 min at 95 °C with a solution of tris(2-carboxyethyl)phosphine/chloroacetamide (TCEP/CAA). The protocol proposed in this study offers to work on minute samples (1 mg of sample or less) and overcame the challenge of separating the proteins from their archaeological matrix. It offers interesting perspectives for archaeological sites or objects where clothing are suspected but hardly detectable, such as burial sites.

Creation of a peptide database of corneous beta-proteins of marine turtles for the identification of tortoiseshell: archaeological combs as case study.

Tortoiseshell is a proteinaceous material derived from the scutes of marine turtles, and was shaped into an abundance of objects, especially luxurious items, at its peak in the seventeenth and eighteenth century. It has continued to be used even after the advent of plastics and remains one of the main causes of illegal poaching of marine turtles, in particular the hawksbill turtle Eretmochelys imbricata. Tortoiseshell is made of structural proteins, of which the most abundant are known as ß-keratins, or 'corneous beta-proteins' (CBPs), a family of short proteins containing a central structure in ß-sheets. There are, however, few CBP sequences of marine turtles in protein databases. The scutes of the five main species of marine turtles (Chelonia mydas, Caretta caretta, Eretmochelys imbricata, Lepidochelys olivacea and Lepidochelys kempii) were analysed by proteomics, using nano-liquid chromatography-Orbitrap-mass spectrometry to generate peptidic markers for species identification. A total of 187 marker sequences were identified, the large majority of them obtained from automated de novo sequencing. The sequences were classified into peptides A to F: A to D at the N-terminus and central region that forms the ß-pleated sheets, E1-4 for a variable region of glycine-repeats region and F at the C-terminus. The markers were tested against a set of combs discovered in various archaeological sites of modern period in France, successfully identifying hawksbill turtle and highlighting patterns of degradation in archaeological tortoiseshell.

Identification of protein remains in archaeological potsherds by proteomics.

We demonstrate here the possibility of identifying proteins trapped in few milligrams of the clay matrix of a 1200-1400 AD Iñupiat 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% trifluoroacetic acid, digestion with excess of trypsin, nanoLC, nanoESI FT-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 Iñupiat 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.

Molecular markers in keratins from Mysticeti whales for species identification of baleen in museum and archaeological collections.

Baleen has been harvested by indigenous people for thousands of years, as well as collected by whalers as an additional product of commercial whaling in modern times. Baleen refers to the food-filtering system of Mysticeti whales; a full baleen rack consists of dozens of plates of a tough and flexible keratinous material that terminate in bristles. Due to its properties, baleen was a valuable raw material used in a wide range of artefacts, from implements to clothing. Baleen is not widely used today, however, analyses of this biomolecular tissue have the potential to contribute to conservation efforts, studies of genetic diversity and a better understanding of the exploitation and use of Mysticeti whales in past and recent times. Fortunately, baleen is present in abundance in museum natural history collections. However, it is often difficult or impossible to make a species identification of manufactured or old baleen. Here, we propose a new tool for biomolecular identification of baleen based on its main structural component alpha-keratin (the same protein that makes up hair and fingernails). With the exception of minke whales, alpha-keratin sequences are not yet known for baleen whales. We therefore used peptide mass fingerprinting to determine peptidic profiles in well documented baleen and evaluated the possibility of using this technique to differentiate species in baleen samples that are not adequately identified or are unidentified. We examined baleen from ten different species of whales and determined molecular markers for each species, including species-specific markers. In the case of the Bryde's whales, differences between specimens suggest distinct species or sub-species, consistent with the complex phylogeny of the species. Finally, the methodology was applied to 29 fragments of baleen excavated from archaeological sites in Labrador, Canada (representing 1500 years of whale use by prehistoric people), demonstrating a dominance of bowhead whale (Balaena mysticetus) in the archaeological assemblage and the successful application of the peptide mass fingerprinting technique to identify the species of whale in unidentified and partially degraded samples.

Identification of the earliest collagen- and plant-based coatings from Neolithic artefacts (Nahal Hemar cave, Israel).

Mortuary practices in human evolution record cognitive, social changes and technological innovations. The Neolithic Revolution in the Levant was a watershed in this domain that has long fascinated the archaeological community. Plaster modelled skulls are well known at Jericho and several other Neolithic sites, and in Nahal Hemar cave (Israel, ca. 8200 -7300 cal. BC) excavations yielded six unique human skulls covered with a black organic coating applied in a net pattern evoking a headdress. This small cave was used as storage for paraphernalia in the semi-arid area of the Judean desert and the dry conditions preserved other artefacts such as baskets coated with a similar dark substance. While previous analysis had revealed the presence of amino acids consistent with a collagen signature, in the present report, specific biomarkers were characterised using combined proteomic and lipid approaches. Basket samples yielded collagen and blood proteins of bovine origin (Bos genus) and a large sequence coverage of a plant protein charybdin (Charybdis genus). The skull residue samples were dominated by benzoate and cinnamate derivatives and triterpenes consistent with a styrax-type resin (Styrax officinalis), thus providing the earliest known evidence of an odoriferous plant resin used in combination with an animal product.

Proteomic profiling of the photo-oxidation of silk fibroin: implications for historic tin-weighted silk.

The stability of silk proteins to ultraviolet light is an issue of significant concern in both the appearance retention of silk-derived products and the preservation of historic silk textiles. Until now, evaluation of silk degradation has only been performed at the holistic, rather than molecular level. This article describes the first proteomic profiling of silk photo-oxidation, characterizing protein primary level modification leading to coloration changes, and evaluating the effects of tin weighting on photodegradation. Heavy-chain fibroin, the main proteinaceous component of the silk thread, is a repetitive, highly crystalline protein with a content rich in tyrosine. Photoproducts of tyrosine were characterized and the levels of oxidative modification at the protein primary structural level correlated with changes in coloration and tensile strength. The effect of tin as a weighting agent used on historical fabrics was examined. Tin-weighted fabrics were evaluated following two treatments (pink and dynamite) and proteomic analysis revealed a significant increase in oxidatively modified amino acid residues within the pink-treated silk. These findings offer new insight into the molecular-level oxidation of silk proteins under UV exposure, and the effects of silk treatments in either exacerbating or ameliorating this degradation.

Mineralization of the metre-long biosilica structures of glass sponges is templated on hydroxylated collagen.

The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

Comment on "Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry".

We used authentication tests developed for ancient DNA to evaluate claims by Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon samples pass these tests, absence of amino acid composition data, lack of evidence for peptide deamidation, and association of alpha1(I) collagen sequences with amphibians rather than birds suggest that T. rex does not.