Proteins in Art, Archaeology, and Paleontology ... - ACS Publications

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Proteins in Art, Archaeology, and Paleontology: From Detection to Identification Sophie Dallongeville,† Nicolas Garnier,‡ Christian Rolando,† and Caroline Tokarski*,† †

Miniaturisation pour la Synthèse, l’Analyse & la Protéomique (MSAP), USR CNRS 3290, Université de Lille 1 Sciences et Technologies, 59655 Villeneuve d’Ascq Cedex, France ‡ SARL Laboratoire Nicolas Garnier, 63270 Vic le Comte, France 3.3.2. Pyrolysis Gas Chromatography and Pyrolysis Gas Chromatography−Mass Spectrometry Applied to Ancient Samples 3.4. Alternative Techniques for the Analysis of Amino Acids or Low Molecular Weight Tags for Protein or Binder Characterizations in Ancient Samples 3.4.1. Capillary Zone Electrophoresis Applied to the Study of Ancient Samples 3.4.2. Inductively Coupled Plasma Mass Spectrometry Applied to Artwork Binder Characterization 4. Protein Identification Based on Immunological Methods: Application to Ancient Samples 4.1. Gel Immuno-Based Techniques Applied to the Study of Ancient Samples: Gel Immunodiffusion, Western Blot, and Alternative Techniques 4.2. Radioimmunoassay for Protein Detection in Fossils and Archeological Materials 4.3. Enzyme Immunoassay and Enzyme Linked Immunosorbent Assay Technique for Protein Identification in Art, Archeology, and Paleontology 4.3.1. Enzyme Immunoassay and Enzyme Linked Immunosorbent Assay and Derived Techniques Applied to Archeological and Fossil Materials 4.3.2. Enzyme Linked Immunosorbent Assay Applied to Paint Media Analysis 4.4. Immunofluorescence Applied to Ancient Samples 4.5. Chemiluminescence for the Study of Ancient Samples 4.6. Immuno-Surface-Enhanced Raman Scattering for Painting Analysis and Related Techniques 4.7. Electrochemical Immunoassay Applied to Artwork 5. Proteomic-Based Methodologies and Related Mass Spectrometry-Based Approaches Applied to Paleontological, Archaeological, and Artwork Samples

CONTENTS 1. Introduction and Scope 2. Detection and Localization of Proteins in Ancient Samples 2.1. Staining Methods for Protein Detection in Paint Cross-Sections and Fossil Samples 2.1.1. Paint Cross-Section Staining Using Visible Dyes 2.1.2. Paint Cross-Section Staining Using Fluorescent Dyes 2.1.3. Staining Method Applied to Fossils and Archeological Samples 2.2. Spectroscopic Techniques Applied to Paleontological, Archeological, and Art Samples 2.2.1. Infrared Spectroscopy and Raman Spectroscopy Applied to Ancient Samples 2.2.2. Fluorescence Spectroscopy Applied to Paint Analysis 2.2.3. Electron Paramagnetic Resonance Applied To Study Protein Environment and Interactions 2.3. Time of Flight Secondary Ion Mass Spectrometry Imaging Applied to Ancient Samples 3. Protein Identification Based on Amino Acid Composition: Application to Cultural Heritage Samples 3.1. Chromatography-Based Techniques Applied to the Study of Amino Acids in Fossils 3.2. Analysis of Amino Acids from Artwork Using Paper Chromatography and Liquid Chromatography 3.3. Analysis of Amino Acids from Artwork Using Gas Chromatography-Based Techniques 3.3.1. Gas Chromatography Applied to Ancient Samples

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22 Received: January 20, 2015 Published: December 28, 2015

© 2015 American Chemical Society

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DOI: 10.1021/acs.chemrev.5b00037 Chem. Rev. 2016, 116, 2−79

Chemical Reviews 5.1. Peptide Mass Fingerprint for the Identification of Proteinaceous Binders in Artwork 5.2. Tandem Mass Spectrometry Applied to Artwork Samples 5.3. Proteomics in Archeology and Paleontology 5.3.1. Identification and Sequencing of Proteins from Fossils and Ancient Bones (Paleoproteomics) 5.3.2. Identification of Proteins and Protein Residues in Archeological Samples 5.4. Current Challenges of Proteomics Analysis in Paleontological, Archeological, and Artistic Samples 5.4.1. Study of Protein Modifications in Ancient Samples Using Bottom Up Proteomic Approach 5.4.2. Toward Intact Protein Analysis and Top Down Proteomics To Monitor Protein Structure in Ancient Samples from Cultural Heritage 6. Conclusion Author Information Corresponding Author Notes Biographies Acknowledgments Abbreviations References

Review

advances in analytical chemistry allowed the emergence of new and more efficient methods for the study of ancient proteins. We chose to build this Review by providing a technical evolution of analytical methods and tools that, while evolving, helped to overcome barriers in the knowledge of studied samples. First, we focus on protein localization techniques and on protein detection methods. We discuss the stains that were mostly used for the analyses of paintings, fossils, and archeological objects, as well as the newest ones with higher sensitivity such as fluorescent stains derived from proteomics. Another main focus is given to spectroscopic techniques such as infrared and Raman vibrational techniques applied to paleontological, archeological, and art samples. The most recent applications are detailed such as EPR experiments for the study of proteins in their environments. Imaging techniques are also considered as synchrotron-based imaging or TOF-SIMS experiments. In the second part, we describe the most often used methods for protein identification based on amino acid composition (e.g., GC, HPLC coupled or not to MS, Edman sequencing, CZE). We discuss the limitations of these techniques such as the loss of information on biological origins of proteins or their chemical modifications. In the third part, advantages of immunological techniques applied to Cultural Heritage are shown; in particular, new localization/identification methodologies inspired from biological methods such as ELISA or SERS nanotags are presented. In the last part, we describe proteomic methodologies that allow a complete identification and characterization of proteins. We show how proteomics has recently been applied to a broad range of ancient samples from Renaissance paintings to dinosaur bones. In particular, we explain the two main strategies proposed until now for the identification of proteins, that is, peptide mass fingerprinting and peptide sequencing using tandem mass spectrometry. We show how these strategies are applied to art, archeological, and paleontological samples, and we explain advantages and the power offered by proteomics in comparison with other classical techniques. Limitations and new methodological developments related to specific questions such as the protein biological origin or chemical modifications are presented and discussed. Finally, the newest challenges of proteomics analysis in paleontological, archeological, and artistic samples are presented.

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1. INTRODUCTION AND SCOPE Paleontology, archeology, and art history provide crucial information for the understanding of human evolution throughout ages and its habits. Past animal and plant species are also studied by paleontology and archeology. Paleontology studies traces of life prior to the Holocene Epoch (11 700 BP). Archeology focuses on human activities and evolution from the stone age prehistoric period (3.5 million years BP). Art history studies art objects from their development to their stylistic contexts. In these different fields, proteins are key molecules. In paleontology, protein sequences from fossils of extinct species contribute to reveal evolutionary links. In archeology, bone proteins may offer information for physiological studies. In the case of archeological objects, the identification of proteins constitutive of an object (e.g., textiles), detected on an object surface (e.g., stone tools) or impregnated in an object (e.g., ceramics), reveals human habits from the past and commercial exchanges. In artwork, proteins were widely used as painting binders, as adhesives for gildings, or they were included in mortars and paint grounds. The identification of protein-based material is important information to fully understand the manufacture process; it informs of the technique used by an artist, and therefore it provides essential information for art historians. It may also support the choice of the most appropriate conservation conditions or restoration procedures. First examinations of artwork materials were described in several treatises dated from the beginning of the twelfth century. Since the 1930s, various methods have been developed to study proteins of Cultural Heritage samples. One of the main difficulties encountered by scientists is to find a satisfying method for chemical analysis, despite the small sample amount available and considering the complex matrix that traps the proteins. In this Review, we intend to show how technical

2. DETECTION AND LOCALIZATION OF PROTEINS IN ANCIENT SAMPLES Materials used in the past in artworks and crafts have been the subject of numerous investigations. Human beings demonstrated particular interest in understanding the recipes used by their ancestors to realize masterpieces that lasted throughout centuries. In particular, ancient treatises dealing with raw materials and painting techniques show evidence of the importance of arts and conceptions of artwork in the society.1,2 For example, De Diversis Artibus of Theophilus Presbyter, written in the twelfth century, emphasizes the techniques used in applied arts in high Middle Ages.3 In particular, the recipes of pigment mixtures providing particular hues are exposed as well as advice for making glue and gilding. Dating from the 15th century, Cennino Cennini’s treatise Il libro dell’arte4 deals with the art during the Renaissance period. Several practical directions are given for the preparation of colors, grounds, and glues. In particular, fresco and secco techniques are discussed but also distemper and oil-based techniques. The knowledge of the great Master Leonardo Da Vinci concerning the art of painting is also described in the Trattato della pittura (the first treatise 3


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presence of peptide bonds in paint samples; the experiment is based on the complexation of copper(II) ion with the amide groups (violet color obtained); (iii) the Millon reaction to detect tyrosine; the formation of a mercury complex is formed following activation of the tyrosine with nitric acid (salmon rose color obtained); and (iv) the Sakaguchi reaction to detect the presence of arginine using 1-naphthol and sodium hypobromite (red-salmon rose colored complex obtained). Ponceau S was also proposed.16 It stains egg yolk proteins orange-red, and it stains glue proteins pink-red. Another dye called Coomassie Brilliant Blue R-250 that could be used without acidic solution was often preferred.16 It forms strong but noncovalent complexes with proteins via hydrophobic reactions, hydrogen bonds, and electrostatic interactions between the dye sulfonate group and the protein ammonium residues or basic amino acids.16 With similar chemical structure as compared to the Coomassie Brilliant Blue R-250, amido black was introduced in 1977.17 It allows the discrimination between egg and glue using a single staining experiment. Amido black reacts specifically with egg proteins at pH 2 and with collagen proteins at pH 7, while at pH 3.6 all proteins are indistinctly stained. This staining specificity can be explained by the amino acid compositions of the proteins. As egg white and egg yolk have high concentrations of amino acids with acidic side chains (Asp, Glu) contrary to gelatin, at pH 2 the majority of acid functions are protonated (COOH) (i.e., the protein−dye interaction is not disturbed). On the contrary, at pH 7 acid functions are ionized (COO−), and a strong repulsion occurred with the sulfonate groups (SO3−) of the dye. In the case of gelatin, due to the high content of hydrophobic amino acids, the protein−dye complex is stabilized by hydrophobic interactions, thus allowing the staining. Nevertheless, the critical point of these staining approaches lies in the fact that all of these molecules are visible stains, and, according to the color of the pigment present in the investigated layer, the staining can be difficult or impossible to discern. Additionally, these methodologies based on visible stains suffer from limited sensitivity. 2.1.2. Paint Cross-Section Staining Using Fluorescent Dyes. In the late 1980s, the application of fluorescent dyes to the study of proteins in paintings was proposed.18 In particular, the uses of fluorescamine, LISSA, and FITC were described. These compounds react with primary amines of proteins (and also sulfhydryl groups on proteins for FITC) to form fluorescent products resulting in blue color for fluorescamine (excitation wavelength 390 nm, emission wavelength 465 nm, 475 nm), yellow-red color for LISSA (excitation wavelength 570 nm, emission wavelength 590 nm), and green color for FITC (excitation wavelength 495 nm, emission wavelength 519 nm). DC-C7A fluorochrome was also studied.19 Dansyl chloride reacts with primary amino groups in aliphatic and aromatic amines to produce sulfonamide adducts; cycloheptaamylose (cyclodextrin) allows the complex to be soluble in water. By comparison with other fluorescent stains commonly used (e.g., LISSA, FITC), DC-C7A appeared to be more efficient for the staining of casein-based paint media. On the other hand, the experimental drawback is that it did not stain egg white significantly. Recently, a new ruthenium complex-based stain available under the commercial name of SYPRO Ruby was proposed to map proteins in ancient samples.20−24 This fluorescent stain (bathophenanthroline disulfonate ruthenium complex; excitation wavelengths 280 and 450 nm, emission wavelength 610 nm) is usually employed for the detection of proteins separated by gel electrophoresis.25 Its use was

Codex Urbinas Latinus 1270 preserved in the Library of the Dukes of Urbino and then the Vatican).5 The treatise argues that painting is a science. Beyond technical aspects of the application of the perspective rules, light and shadow, proportion or movement, the treatise exposes good material mixtures allowing the desired effect and the stability of the masterpiece.5 At the beginning of the twentieth century, several chemists such as Wilhelm Ostwald and Arthur Pillans Laurie studied the properties of painting components.6−8 They insisted that it is necessary for a painter to understand why some pigments are incompatible, or why a painting made with a specific medium will be stable when the same pigment mixed with another medium will not last. The chemistry of paints9,10 was addressed to craftsmen or painters to help them understand the properties of the materials they used (pigments, media) and the right way to use these materials (e.g., the stability of several formulations was evaluated). However, it was admitted that some materials such as gelatin were complex mixtures and that they were not yet understood even by chemists. Since this time, chemical experiments were undertaken to understand the properties of paintings in terms of time resistance, color changes, and degradation (crackling, yellowing...). Before investigating and understanding degradation phenomena, it became essential to identify the compounds that have been used by the artist. In 1904, Ostwald mentioned that “the pigments are the same in all the processes of painting, however different they may look in the finishes work, and only the binding medium is different.”6 This sentence demonstrates the importance of identifying the binding medium during the study of a painting. 2.1. Staining Methods for Protein Detection in Paint Cross-Sections and Fossil Samples

2.1.1. Paint Cross-Section Staining Using Visible Dyes. The pioneer scientific experiments referring to proteins in artwork were based on staining methods. Ostwald was the initiator in this domain.11 In 1936, he used biological dyes such as iodesine or acid green to stain proteins in painting crosssections. These stains bind to proteins through electrostatic and hydrophobic interactions. Ostwald proposed the use of optical magnification to distinguish the different layers and to observe the effect of the stain on them. The discrimination of some binders was for the first time possible. For example, gelatin and casein were observed in situ with acid green, whereas only glue was stained with an ammonia solution of iodesine. Subsequently, the preparation of paint cross-section was optimized by the introduction of synthetic resin (methyl methacrylate) for embedding them.12 The replacement of microtome cutting by polishing allowed the preparation of friable samples.12 A set of microchemical tests was applied to Flemish paints;13 in particular, siccative oil, tempera (egg white), and animal glue were discriminated with success. For example, tempera and glue paints were stained red with a solution of ammonium hydroxide containing iodesine; they were also stained violet using an aqueous solution of methyl violet, whereas oil paints were not stained. Another test involving vanillin did not stain the glue samples, but it stained the tempera paints red/violet. Applied to the study of paint samples embedded in cold setting polyester resin, acid fuchsin was shown to be an effective stain of protein binders (i.e., reaction with ammonium groups of proteins).14 The detection of proteins in paintings was also performed using a set of colorimetric reactions15 including (i) the use of ninhydrin that reacts with the amine groups of proteins (roseviolet color obtained); (ii) the Biuret reaction to detect the 4


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based on the peroxidase activity of the hemoglobin that cleaves oxygen molecules from hydroperoxide and catalyzes the reaction from 3,3′,5,5′-tetramethylbenzidine (colorless-light yellow) to its oxidized form (orange to green; blue for very high concentrations of blood). The Hemastix protocol was optimized to avoid positive reactions to other compounds. In particular, a treatment with EDTA chelating agent has shown to reduce false positive with no loss of sensitivity to hemoglobin detection.31 This optimized protocol was successfully tested on fluted projectile points from Eastern Beringia (these fluted points are tangible remains of the first Native Americans who crossed the Bering Land Bridge).31 Recently, this method was applied to a wide range of modern samples (plant, animal, manufactured substances, metals, and chemical solutions), blood residues on slides (5−15 years old), and experimental stone artifacts, to demonstrate the specificity and the reproducibility of the test.32 As initially suggested by Loy and co-workers,31 Hemastix provides a starting point for hemoglobin identification in a screening approach, but results have to be confirmed by complementary analyses.32

considered for the mapping of proteinaceous binder due to the straightforward staining procedure, which consists of the application of a droplet directly on the cross-section surface using a Pasteur pipet. Microspectrofluorometry is used as the detection method. For example, applied to the localization of proteins in a complex multilayered structure such as polychromy,22 this fluorescent stain was able to localize a thin proteinaceous layer between the silver leaf and the lacquer as shown in Figure 1. The presence of this thin layer in the

Figure 1. (A) Microscopic view (×500) of the polychrome sample cross-section under visible light. The layers numbered 1, 2, and 3 correspond to the ground, the bole, and the green glaze, respectively. (B) Microscopic view (×500) of the polychrome sample cross-section after Sypro Ruby staining using Leica filter D. The fluorescence signal emitted by the proteins is red/orange and shows the presence of a thin proteinaceous layer on the silver leaf and proteins in the ground layer. Reprinted with permission from ref 22. Copyright 2013 Royal Society of Chemistry.

2.2. Spectroscopic Techniques Applied to Paleontological, Archeological, and Art Samples

2.2.1. Infrared Spectroscopy and Raman Spectroscopy Applied to Ancient Samples. 2.2.1.1. Infrared and Raman Techniques Applied to Artwork Samples. Since the beginning of analytical studies applied to Cultural Heritage samples, spectroscopic techniques and, in particular, vibrational spectroscopy techniques such as IR spectroscopy33,34 and Raman spectroscopy35,36 represent relevant methods to obtain simultaneous molecular information on mineral and organic phases. Both techniques deal with the energies associated with vibrational motions between atoms in a molecule. In IR spectroscopy, the frequencies absorbed by a sample positioned in the path of an IR beam are measured (transmission or reflection mode), while the Raman technique consists of measuring the light scattered by a sample previously excited with a laser. 2.2.1.1.1. Infrared Spectroscopy Applied to Artwork. In the mid 1950s, the first application of IR spectroscopy to Cultural Heritage samples focused on characterization of dammar, mastic, and resins from paintings.37 Analysis of proteins, in a complementary way to the study of other organic components from ancient paints (e.g., polysaccharides, oils, waxes), was performed by IR spectroscopy from the end of the 1960s.38 Paint samples (few milligrams) were dissolved in water containing mineral salts, and the resulting mixture was dried before IR analysis. Applied to the analysis of paintings from the XVth− XVIth centuries, the technique allowed the detection of CO and NH stretchings from the amide peptidic bonds, respectively, at 1640 and 1540 cm−1. The presence of proteins was also confirmed by chromatographic techniques that allowed the identification of amino acids. With an enhanced sensitivity, FTIR was applied to microgram quantities of samples.39 Each sample (sampled from paintings or protein-based films) was ground in anhydrous potassium bromide and pressed under vacuum to form a pellet that is mounted at the focal point of the beam condenser (transmission measurements). Characteristic bands of egg yolk proteins were observed on the IR spectrum (Figure 2A) despite the difficulties caused by the presence of linseed oil (triglyceride fatty ester features) and/or pigments. In particular, five bands were pointed out: the NH stretch at 3289 cm−1, the amide II overtone at 3080 cm−1, the amide I band including CO

multilayered structure is described as being involved in the bright luminous effects of the polychromy. The SYPRO Ruby, used for protein mapping, is generally used in combination with other analytical techniques such as mass spectrometry to allow complete characterization of the protein media;22 for example, studies of XVIIth polychrome object have identified glue proteins from different biological origins,22 and studies of XVII−XVIIIth centuries gildings and a painting of the main altarpiece of Miranda do Douro Cathedral in Portugal have shown different proteins in samples (see details in the proteomics section).23 2.1.3. Staining Method Applied to Fossils and Archeological Samples. The staining of proteins in fossils and archeological samples has been described since the early 1980s.26,27 For example, mercurochrome was used on fossil teeth (transversal and longitudinal planes obtained using a wire string saw or abrasive paper) embedded in an epoxy resin (the stain reacts with sulfhydryl and disulfide groups of proteins).28,29 Studies were performed using fluorescence microscopy analysis, and the compound exhibited an orange fluorescence (excitation wavelength 470 nm, emission wavelength 500−530 nm). Staining of fossil teeth was most pronounced near the pulp cavity, showing the presence of proteins in the dentin.26 Colorimetric tests based on the use of ninhydrin27 (detailed previously) or OPA27 were also applied to detect the presence of proteins on prehistoric tools. In the presence of βmercaptoethanol, OPA reacts with primary amine groups of proteins to form a blue colored fluorescent product (the unbound dye is nonfluorescent) (excitation wavelength 340 nm, emission wavelength 455 nm). In the late 1990s, Hemastix was applied to detect the presence of blood residues on stone artifacts from the archeological excavation of Ç ayönü Tepasi in Turkey.30 It is a colorimetric test strip originally designed to detect blood in urine. It contains diisopropylbenzene dihydroperoxide and 3,3′,5,5′-tetramethylbenzidine. The test is 5


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Figure 2. (A) (a) FTIR spectral features obtained at 25 °C for a freshly prepared film of egg yolk. (b) A KBr micropellet spectrum of a naturally aged egg yolk film from the Fogg test plates collection. (c) A KBr micropellet FTIR spectrum of a naturally aged film of egg yolk and lead white from the Fogg test plates collection. Insets show an expanded view of the carbonyl spectral features. (B) (a) Paint sample from a wood panel by Guariento di Arpo depicting a Principatus Angel, painted about 1350. (b,c) Paint samples from a wood panel probably by Antonio da Saliba, depicting the Enthroned Madonna, painted in the last quarter of the 15th century. The white sample (b) is from the base of the Madonna’s throne, while the red sample (c) is madder glaze from the Madonna’s robe. Reprinted with permission from ref 39. Copyright 1990 Maney Publishing.

stretching at 1654 cm−1 and NH2 deformation at 1632 cm−1, and the amide II band at 1542 cm−1. The amide features were clearly present in spectra of fresh egg yolk films (Figure 2Aa) and egg yolk films naturally aged 50 years (Figure 2Ab). The introduction of lead white in egg yolk film resulted in a splitting of the amide II band into two components at 1542 and 1515 cm−1 (Figure 2Ac). This splitting, observed in fresh samples and

in samples of 50 years old, was assigned to a change in the protein structure. The bands corresponding to the NH stretching, amide I, amide II, and amide II overtone were identified during the analysis of painting samples of museum collections (Figure 2B) (e.g., Principatus Angel painted by Guariento di Arpo in the XIVth century from the Fogg Art 6


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Figure 3. μFTIR analysis of a pressed painting fragment from Foladi site. Visible light picture, average FTIR spectra, and chemical mappings. Regions of interest selected to calculate maps are indicated in wavenumbers. Map size, 144 × 384 μm2; step and beam size, 12 × 12 μm2. Reprinted with permission from ref 50. Copyright 2008 The Royal Society of Chemistry.

required. The first application to the study of binding media41 using FTIR microscopy in transmission mode proposed to cover each cross-section by silver chloride, and then the sample was gently pressed and the thickness was made uniform with a microtome. This technique was successfully applied to the study of several easel paintings from the XVth to XVIIIth centuries. To classify the different binding media with or without interactions with other paint components, chemometrics methods such as HCA,42 PCA,42−44 and KNN42 were applied to FTIR data. In the early 2000s, imaging experiments based on reflection FTIR analysis were proposed for the study of painting crosssections.45 Several independent detectors (FPA) and a step-scan interferometer were used. The spatial resolution determined by the magnication of the microscope and the pixel size in the FPA was able to reach 6−7 μm (the paint layer thickness range is generally 1−50 μm). Each cross-section was embedded in a polyester casting resin, and the sample was then polished. Information on the distribution of the organic functional groups in the individual layers of the studied cross-sections was shown; in particular, the study of a Rembrandt cross-section was successfully achieved.45

Museum). These results have demonstrated that FTIR spectroscopy is a valuable tool for protein detection in art samples. These characteristic spectral features were used in combination with complementary analytical techniques (e.g., differential scanning calorimetry) for the study of complex binding media of several paints from the 16th centrury.40 For example, a medium based on oil and egg was distinguished from an oil-based medium. Another example showed that different regions of a XIXth century painting were formulated with different organic components (e.g., drying oil; oil−resin mixture; proteinaceous material−wax mixture).40 With the introduction of infrared microscopes, it became possible to study cross-sections and therefore localize the chemical information across the layer structure of a paint sample.41 Thus, the spatial resolution of microscopes (refers to the size of the smallest feature detectable) represents an important step forward. Using a single-element MCT detector, the spatial resolution is related to the IR beam aperture dimension, which is limited by the theoretical diffraction limit (∼10 μm).34 To obtain enough energy to provide spectra with good signal-to-noise ratios, a minimal aperture of 20 × 20 μm is 7


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Figure 4. Visible (a) and five chemical (b−f) images collected from a painting cross-section. The cross-section contains two layers: a top layer of red paint over a white ground. In (a), the top layer of red paint is visible as a diagonal band running from the upper left to the lower right; the embedding medium is in the upper right (appears colorless), and the ground is the mottled gray region in the lower left of the image. The chemical images correspond to integration under: (b) 1585−1592 cm−1, suggestive of the red organic colorant alizarin; (c) 2850−2858 cm−1, CH stretches, likely stemming from egg yolk present as a paint binder; (d) 1493−1547 cm−1, amide II stretches, suggesting proteinaceous materials (egg yolk and animal glue in the paint and ground layers, respectively); (e) 1400−1446 cm−1, carbonate n3 asymmetric stretches in calcite, probably used as a laking substrate for the organic colorant; and (f) 1076−1199 cm−1, sulfate n3 asymmetric stretches in gypsum, used as a ground. For color interpretation details, refer directly to the discussed article. Reprinted with permission from ref 52. Copyright 2012 Elsevier.

Recently, multiple-beam synchrotron-based mid-infrared imaging was applied to the study of painting cross-sections.52 Twelve collimated beams from the synchrotron were combined into a bundle of beams that is refocused onto the sample plane of the infrared microscope.53,54 The instrument covers more than twice the sampling area in a shorter time with 100 times better spatial resolution as compared to single synchrotron beam systems (operating at ∼5 × 5 μm2). Applied to the study of cross-sections, the mapping showed different inorganic and organic components in a paint layer 10 μm-thick (Figure 4).52 NIR reflectance imaging spectroscopy (800−2500 nm range; 4000−14 000 cm−1) was also evaluated for mapping and identification of proteinaceous binders in artwork.55−57 The spectral features are mainly related to the vibrational overtones and combination bands of fundamental absorptions. In particular, the NIR vibrational features of proteins are related to the CO, NH, and CH2 groups.58 The features of binding media in NIR are weaker and less specific than their corresponding signatures in MID (2500−25 000 nm range, 400−4000 cm−1); however, they are less affected by the interferences that may be caused by pigments.55,56 Hyperspectral NIR imaging instruments provide resolutions down to 5−10 nm, and a 10 μm thick sample can be analyzed.55,59 Applied to the study of binders of a manuscript dated from the XVth century, NIR reflectance imaging spectroscopy combined with FORS experiments (spectral data obtained from the UV−visible to the short-wave IR regions, providing information on pigments and on the functional groups such as CH, OH, NH) allowed successful characterization and localization of fat-containing binders.55 For example, the spectral features of egg yolk were shown, using the first overtones of the asymmetric and symmetric stretching of CH2 at 1729 and 1760−1763 nm. NIR spectroscopy imaging applied to the study of a painting dating from the XVth century proved the presence of different binders (animal skin glue, egg yolk, or a mixture of both) localized in different parts of the paint. The results were verified using a complementary analysis (analysis of amino acids using HPLC).56 Another study57 applied to a Renaissance wooden painting and a wall painting dating from the XVth century has proposed the use of relevant chemometric methods to differentiate the different types of proteins (multivariate chemical maps recorded in the 700−7500 cm−1 range). For example, the NH band of animal glue was detected around 4896 cm−1, while the marker bands of egg proteins were shifted to about 4863 cm−1. In addition to the characterization of protein binders and imaging experiments, other essential points were investigated using FTIR spectroscopy, among which are the understanding of

ATR objective (ATR germanium crystals coupled with a focalplane array) was also coupled with an IR microscope to increase spatial resolution. This technique allows the investigation of smaller areas maintaining the same IR beam aperture due to the magnification factor of a crystal with high refractive index that is placed in contact with the sample.34 For example, imaging experiments were conducted on albumen photographs from the XIXth and XXth centuries, and a spatial resolution of ca. 3−4 μm was shown.46 In particular, different protein contents were detected using different absorbance values of amide II band for light and dark regions of the same photograph sample. The technique was also applied to the study of paint samples from the National Gallery of London.47 Stratigraphic analyses of the organic materials in samples of wall paintings from the XIIth and XVIIIth centuries were also successfully provided.48 In particular, the proposed novel technique for sample preparation showed its high capability during analyses of samples containing very low amounts of organic material. The methodology is based on cyclododecane (instead of synthetic embedding resins as polyesters, epoxies, acrylics) as a consolidant and barrier coating to encapsulate the sample. The synchrotron emission in the infrared domain was also used as a source for FTIR microscopy for the study of organic matter in paintings.49,50 The synchrotron radiation brightness is high so it enables the reduction of the beam size below 8 μm without a significant loss of photons (spatial resolution down to 5 μm × 5 μm and may be reached in transmission or reflection). Spatial resolution is thus improved, and the signal-to-noise of spectra is also increased. The micro-SR FTIR technique showed its high capability during the study of a painting dated from the VIIIth century AD from Foladi sites.50 In this study, the spectral quality (Figure 3) was crucial to discriminate the various compounds present in this paint cross-section, in particular, in the CO stretching domain. Thus, it was possible to distinguish ester, acid, amide, carboxylate, carbonate, and oxalate groups. SR FTIR analysis coupled to 2D FTIR imaging with a conventional source was also applied to the study of the finishing technique of an ancient musical instrument (XVIIIth century cello).51 The SR FTIR imaging experiments have identified two layers with different chemical compositions. The spectra of the primary layer showed characteristic features of proteinaceous materials (e.g., OH and NH stretching vibrations, alkyl bands, amide I and II bands), whereas the surface layer showed characteristic bands of aged mixtures of drying oil and natural resins (e.g., broad CO stretching, alkyl stretching). Complementary techniques were used to identify precisely the organic components; for example, the presence of glue was revealed by GC−MS in the protein-containing layer. 8


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Table 1. Tentative Peak Assignments of Raman Spectra Obtained from Thin Films of the Eight Different Protein-Based Media Examineda dairy proteins (Raman shift, cm−1)

collagen (Raman shift, cm−1)

egg yolk

egg white

casein

milk

fish glue

parchment glue

rabbit glue

ox glue

605

602

604 644

600

598

603

598

718 767

698 758

601 632 703

729

739

781 816

781 814

797 815

789 813

856

857

853

852

919 938

919 921

919 938

918 938

1003

1003

1003

1002

1033

1033

1034

1033

763 780

828 849 873

1003

1080 1125

1440 1555 1657 1725 2726 2852

900 934 954 1003

826 852 878 931 954 1003

1032 1041

1033

1125

1127

1207 1241 1336

1206 1248 1338

1401 1448 1550 1605 1660

1410 1449 1551 1616 1665

2729 2873 3061

2870

828 851 876 915 954 1019 1032 1052 1087 1120 1140

1092 1122

1261 1338 1379 1414 1452 1551 1608 1663

1206 1244 1318

1204 1242 1319

1412 1449

1411 1448 1557 1603 1665

2725 2887 2976.5

2882

1607 1665

2880 2985.3

1165 1208 1249 1315 1379

1168 1205 1244 1313

1448 1603 1666

1448 1550 1602 1663

2873

2880

tentative assignmentb amide VI N−H deformation Tyr C−S stretching cysteine/trans and gauche Trp N−H wagging C−C stretching Tyr Tyr Trp C−C vibration C−C stretching C−C vibration, phosphate symmetric stretching Phe ring breathing C−C stretching Phe C−C stretching C−C, C−N stretch C−CT stretch aliphatic CH3antisymmetric; aromatic CH2 rock Phe/Tyr amide III CH2 deformation aspartic and glutamic acids (CO stretching) C−H bending Trp Phe/Tyr amide I CO stretching aliphatic C−H stretching C−H stretching aromatic CH stretching

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28)

a

For band references (numbers), please refer directly to the discussed article. Reprinted with permission from ref 72. Copyright 2007 American Chemical Society. Phe = phenylalanine, Tyr = tyrosine, Trp = tryptophan. Numbers in parentheses have been used to label selected peaks in Figures 2 and 3. bReferences 7, 17, 19, 22, 23, 25, 28, and 32−36. Bands that have not been identified are not assigned but are listed for completeness.

showed that the glue binder alone suffered from strong degradation following UV irradiation, whereas the presence of azurite seems to protect the glue binder against UV degradation.63 FTIR experiments were also used to study protein−pigment interactions in various paint reconstructions. The reference paints were formulated with casein or ovalbumin proteins and various pigments (cinnabar, azurite, calcium carbonate, hematite, and red lead).64,65 FTIR analyses showed that protein−pigment interactions and aging have provoked strong changes of the amide II/amide I ratio (mainly considering ovalbumin samples). The relative decrease of the amide II band as compared to the amide I band was correlated to the formation of intermolecular β sheets. The variations of the amide II/amide I ratio were also linked to protein oxidations and protein−metal interactions. On the basis of FTIR experiments and complementary techniques (thermogravimetry, SEC UV and cold vapor generation atomic fluorescence spectrometry, DSC), these studies64,65 showed different protein degradations such as aggregation, oxidation of amino acid side chains, and hydrolysis of the polypeptide chain in both pure and pigmented paints exposed to aging.

interaction mechanisms occurring between components (e.g., between pigments and binders), but also the study of the effects of aging or environmental factors on proteins. During a study related to the selection of the most appropriate retouching binders for restoration of wall paintings, FTIR spectra of artificially aged proteinaceous binders mixed with pigments have suggested the destruction of peptide bonds during experiments in high relative humidity or following UV light exposure.60 In particular, the 1078 and 1170 cm−1 bands were attributed to the formation of free NH2. Another study focused on the degradation process affected by nonproteinaceous materials such as lipids, terpenic compounds, or inorganic pigments during aging.61 Improved by PCA methodology, the study showed that the most affected IR region was between 2900 and 3600 cm−1; this specific region includes the amide band. The amide band region was also shown to be affected by different durations of UV-aging (100, 500, 1000, and 1500 h of exposition) of samples built with various materials such as gypsum, egg albumin, jersey cow casein, rabbit epidermis collagen, linseed oil, white lead pigment, and/or terpenic resin.62 The data interpretation was performed using PCA chemometric tools. A focused study on blue copper pigment and glue tempera 9


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related to their amino acid compositions. For example, Trp is absent from several collagen proteins, whereas casein or eggbased media contain this amino acid (∼760 cm−1) (Table 1). Likewise, for casein, which does not have disulfide bridges, the peak attributed to C−S stretching is absent in the Raman spectrum. However, it can be noticed that these spectral features are detected in the fingerprint region (200−1800 cm−1), which is generally subjected to interferences from other materials such as inorganic pigments and can make the interpretation of the spectra difficult. Consequently, the C−H stretching region (2700−3200 cm−1), characteristic of organic materials, may be used for discrimination between proteinaceous binders. By performing a multivariate analysis (PCA) on 80 Raman spectra in the C−H stretching range, it was possible to group proteins in different clusters and thus to discriminate them using this nondestructive technique.72 PCA analysis was also used to discriminate tempera model binders mixed with different pigments.80 The effects of aging and/or pigments on protein media were also studied.81−83 Studies of models revealed that aging or interaction with pigments may cause intensity changes, disappearance, or emergence of bands. For example, the analysis of the model sample based on egg white showed a peak at 758 cm−1 attributed to the vibrational mode of Trp (Figure 5).82

Another challenging task that also represents one of the main objectives of the research in conservation science is related to in situ measurements.66,67 New insights in FTIR analysis were recently provided with the introduction of a portable instrument.66,68,69 Portable instruments using mid-FTIR (spectral range 7000−900 cm−1, spectral resolution 4 cm−1, spatial resolution 12 mm2, sampling probe: chalcogenide glass fiber optic) and near-FTIR (spectral range 12 500−4000 cm−1, spectral resolution 4 cm−1, spatial resolution 12 mm2, sampling probe: quartz fiber optic) are proposed.66 For example, the use of a portable mid-FTIR reflectance instrument was applied to the study of a mural painting dating from the XVth century, focusing both on inorganic pigments and on organic binders.66,68 The investigated sample area determined by the probe diameter was about 20 mm2. On the basis of FTIR spectra, a PCA chemometric treatment, and FTIR spectra obtained from model samples, the presence of casein on the painting surface was suggested as well as animal glue as original secco finitures (instead of egg, because egg-lipidic absorption at around 1740 cm−1 was absent). Further studies exploiting chemometric methods to extract relevant information from FTIR spectra recorded were proposed and successfully applied to a Renaissance painting.69 It had also been pointed out how this analytical task was challenging due to the variability and the complexity of infrared reflectance features, for example, several large distortions in the spectrum in band shape and position, which may depend on band intensity, sample concentration, and optical layout. Developments of portable FTIR instruments remain a major challenge,70 and recent instruments showed the capability of a mobile laboratory (MOLAB) equipped with an array of state-of-the-art portable and noninvasive instruments to study objects on site,66 such as portable near- and mid-FTIR that allowed the discrimination of organic binders in several paintings (wall paintings and canvas paintings). 2.2.1.1.2. Raman Spectroscopy Applied to Artwork Samples. Raman spectroscopy represents a technique complementary to IR spectroscopy. The coupling of Raman spectroscopy with microscopy71 in the late 1970s has contributed to expand the use of this technique, especially in the Cultural Heritage field. The micro-Raman technique allows the analysis of samples of sizes less than 5 μm2, providing thus a selective analysis (reduced interferences from adjacent particles).72 Since the mid 1980s, the main applications of micro-Raman are related to pigment identification.73,74 The first applications referring to the analysis of protein materials were proposed in the early 2000s.75,76 As protein-based media suffer from intrinsic fluorescence of proteins,75,77 instrumentation improvements (e.g., FT-Raman) and the application of mathematical methods acting on fluorescence background were proposed.78,79 The fluorescence background may be eliminated by subtracting two Raman spectra acquired with two different excitation laser lines. For example, SERDS (micro-Raman experimental apparatus coupled with a tunable diode laser) and SSRS (shifted spectrum obtained by moving the spectrometer grating) are two described methods.79 The first attempts for the characterization of proteins showed the amide I band (∼1667 cm−1), CH2 scissoring band (∼1450 cm−1), and amide III band (∼1245 cm−1).75 With the help of chemometrics tools, it was shown that Raman spectra of various model binders have exhibited differences allowing their discrimination.72 In particular, due to the presence of fatty acid esters, egg yolk has a characteristic carbonyl vibration at 1725 cm−1 (Table 1). For the other protein-based media (egg white, collagen, animal glue), the differences in the Raman spectra are

Figure 5. Raman spectra of egg white aged either naturally or by exposure to light: a depletion of the band from tryptophan at 758 cm−1 is ascribed to photo-oxidation and indicated by an asterisk. This figure is available in color online at www.interscience.wiley.com/journal/jrs. Reprinted with permission from ref 82. Copyright 2008 John Wiley & Sons.

This peak was not present in spectra of samples exposed to light. This phenomenon was ascribed to a partial oxidation of tryptophan in egg white. Bands associated with aromatic amino acids were affected by aging; however, the identification of binders was possible.81 For example, an accurate examination of the C−H stretching region led to the successful differentiation of egg yolk, milk, and collagen-based media. Recently, the protein C−H stretching region was shown to be affected by the presence of particular pigments.83 For example, tempera model samples based on azurite, lead white, and gypsum showed relevant spectral changes. The complementarity of FT-Raman spectroscopy, FORS, and PCA techniques was recently investigated to identify pigments and binding materials 10


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in simulated medieval paints.84 In particular, the reliable discrimination among protein-based and polysaccharide-based media required combined Raman and FORS data (contrary to oil-based binding media that were directly discriminated using FT-Raman). Raman spectroscopy benefited also from developments of portable instruments,66,85−89 to study various Cultural Heritage materials (e.g., pigments, glasses, polymer-based treatments used for conservation). Considering the study of proteins, an outdoor Raman spectroscopy analysis (instrument equipped with 785 nm diode laser, spectral range 250−2000 cm−1, spectral resolution 8 cm−1) of solid amino acids was proposed showing an unambiguous detection of 13 selected amino acids.90 Recently, an approach based on SERS (see related section for details on SERS) was successfully proposed for the sensitive detection of natural amino acids (L-Arg, L-Phe, L-Met) and a tripeptide (glutathione) adsorbed on silver colloids.91 Concentrations of 10−3 M (L-Arg and L-Phe) and 10−5 M (L-Met) were, for example, detected in dried as well as liquid samples. These results support the optimization of Raman instrumentation and its application to the analysis of amino acids/proteins in Cultural Heritage materials. 2.2.1.2. Infrared and Raman Techniques Applied to Ancient Biomaterials from Paleontological and Archeological Contexts. Both IR and Raman spectroscopic techniques were used to probe the presence of proteins in samples from archeological/paleontological sites and to provide information concerning the degradation of these biomaterials92 and probe the organic contamination.93 In particular, FTIR spectroscopy was used to detect proteins in mummified skin,92 soft tissue,94 hair,95 and ancient bones96,97 by investigating characteristic amide bands. For example, the state of conservation or degradation of human mummified skin was studied (studied sections ∼6 μm thick);92 a focus was given to the outer layer of the skin, the Stratum corneum (ca. 15 μm). In particular, the chemical images obtained with the synchrotron infrared microscope (Figure 6) were studied, and proteins were detected via the amide II band (1585−1484 cm−1) in the most external region (in a depth of less than 20 μm) (Figure 6f). Whereas archeological and modern samples present similar profiles in the most external (∼20 μm) skin region (better preserved), differences were pointed out in deeper parts (e.g., absence of amide bands, broadening and merging of amide I and II bands). Hair cross-sections of mummies were also studied.95,98 Keratins from Egyptian mummies dating back more than 2000 years were successfully characterized using bands resulting from alkyl and amide groups. While the signal of the alkyl groups was constant on the whole map, the amide I signal decreased strongly from the center of the hair to its periphery. These observations have suggested a long-term modification of the protein fraction at the hair periphery via the potential disruption of the polypeptide backbone.95 Applied to the study of hair strands found in the burial attributed to Marie de Bretagne (XVth century),98 FTIR imaging allowed for the studying of the distribution of organic coumponds within hair cross-sections and potential structure modifications (e.g., oxidative degradation of disulfide bridges leading to formation of cysteic acid at 1040 cm−1 and cysteine-S-sulfonate at 1022 cm−1). Overall, the multianalytical approach proposed in this work (synchrotron XRF, μXRF, μFTIR, and μSAXS) has provided a possible scenario that led to the exceptional preservation of these tissues, involving the presence of copper and lead.98

Figure 6. Chemical images of a selected skin region obtained with the synchrotron infrared microscope. (a) Optical image of the cross-section and location of the map, (b) chemical image of the fatty chain distribution (band area between 3000 and 2800 cm−1), (c) chemical image of the solid palmitic chain (peak at 1272 cm−1), (d) chemical image of the CO ester location (peak at 1732 cm−1), (e) chemical image of the CO acid location (peak at 1702 cm−1), (f) chemical image of the amide II band location (area from 1585 to 1484 cm−1), and (g) chemical image of the calcium oxalate location (peak at 1319 cm−1). The size of each map is 220 μm × 140 μm. Reprinted with permission from ref 92. Copyright 2005 Elsevier.

During a study related to bone diagenesis, the preservation of proteins was evaluated in archeological bones depending on the burial environment. Two samples from a unique archeological site but from different local environments (depositional, hydrological, and redox) were studied.97 The IR spectra have shown different preservation states of the organic matter (e.g., weak intensity of the amide I absorption band at ∼1640 cm−1 and absence of amide II and III bands at ∼1550 and 1250 cm−1 respectively, versus intense amide I, II, and III bands). In particular, a geographical zone with a fluctuating hydrological regime showed a low level of organic matter in bones. These samples showed also a high level of porosity (porosity measurements performed with the N2-BET method). Another study on protein modifications induced by diagenesis had shown changes in the secondary structure of type I collagen protein using ATR-FTIR spectroscopy combined with curve-fitting of the amide I and II bands.99 The analytical process proposed was able to link the major structural components of the protein to spectra profiles (e.g., for amide I: band aromatic ring at 1610 cm−1, β-sheets at 1630 and 1678 cm−1, random coils at 1645 cm−1, turns at 1692 cm−1, and α-helix at 1661 cm−1). A decrease of the α-helix percentage and an increase of unordered structures (random coils) were observed, indicating that the bone structure has been modified during burial. To improve the spatial resolution of analysis, synchrotron radiation micro-FTIR (SR FTIR) imaging was proposed for the study of the preservation of archeological bones.100 A spatial resolution below 10 μm was reached versus 50 μm with ATR-FTIR. SR FTIR provided evidence of changes induced by the burial environment and 11


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Figure 7. (a) SR FTIR image of the amide I/phosphate ratio (1660/1095) corresponding to an osteon area of the archeological bone sample AB_CH19nb1 from the Chalain Lake site. (b) SR FTIR image of the collagen cross-links represented by the 1690/1660 ratio corresponding to reducible to nonreducible cross-links of the same osteon. (c) SR FTIR image of the ratio of random coils to α-helix subbands (1645/1660) of the same osteon. (d) SR FTIR image of the carbonate/phosphate ratio (1415/1095) of the same osteon. (e) SR FTIR image of the apatite crystallinity (1030/ 1020) of the same osteon. A color scale from red (high intensity) to blue (low intensity) was adopted for all images. Reprinted with permission from ref 100. Copyright 2010 Springer.

distribution of the collagen content in the osteon structure of the archeological bone (Figure 7a).100 Collagen cross-links were represented by the 1690 cm−1/1660 cm−1 ratio (Figure 7b) and the ratio of random coils to α-helix subbands (1645 cm−1/1660 cm−1) (Figure 7c). These features allowed one to provide an image of the collagen structure around the osteon, and more

allowed the localization of altered areas (Figure 7). As an alternative to the curve fitting of absorption bands in each spectrum, absorbance intensities and intensity ratios of particular wavelengths were used to represent the spatial distribution of bone components. As an example, the amide I/phosphate ratio (1660 cm −1 /1095 cm−1 ) was selected to measure the 12


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1217 cm−1) bands were shown in buried sample spectra. A potential explanation lies on the fact that during burial, the mineral phase is partially lost, exposing thus the collagen to hydrolysis. It can be noted that two new shoulder peaks (2852 and 1295 cm−1) were detected in buried samples; they were assigned to CH2 and CH vibrations of lipids and phospholipids from bones. Additionally, the amide I band had also shifted from 1668 to 1656 cm−1 characterizing thus potential structural changes. Raman spectroscopy was also used to evaluate alterations in bone composition.112 Recently, the collagen diagenesis was studied using freshly cut cross-sections, and multivariate statistics for screening purposes.113 The collagen quality was successfully predicted using the ratio between 960 cm−1 (PO stretching band) and 1636 cm−1 (amide I carbonyl stretching band). Skin tissue of mummified remains was also studied using Raman spectroscopy. For example, studies of the late-Neolithic man (5200 years BC) found in a glacier in Oetzal (Iceman)114,115 showed the reduction in the vibrational intensity of protein bands (e.g., amide I and III bands) in comparison to the contemporary tissue, pointing out the degradation of protein components. The study of mummified penguin tissue116 also revealed the degradation of proteins in the fur, claws, skin, and feathers. For example, the amide I stretching region showed two bands of medium intensities at 1670 and 1602 cm−1 instead of a unique medium intensity band at 1665 cm−1 characteristic of helical protein chain conformation. The study of mummified skins of the Alpine Iceman, the Qilakitsoq Greenland mummies, and the Chiribaya mummies from Peru117 showed also the loss of proteins or changes in the secondary protein structure using amide I and III bands. These amide I and III bands were also used to probe the protein presence in skin samples from Egyptian mummies (ca. 2000 BC).118 This study revealed the strongest degradation of organic compounds when sodium sulfate was also detected in the samples; sodium sulfate was attributed to the embalming process of the body that was entirely covered with natron (natural compound composed of sodium carbonate and hydrogen carbonate, sodium sulfate, and sodium chloride). Parchments and vellum (parchement of higher quality) were also studied with Raman spectroscopy.119 These materials are made with animal skins, usually untanned skins of sheep or goat for parchements and untanned skins of lambs, pigs, and young calves for vellum. For a long-term preservation, the animal skins were usually treated with salt-based solutions (sodium or potassium chlorides, ammonium chloride or sulfate, sodium sulphite) using lime and potash alum. The animal skins were also treated with flour, egg yolk, or natural oil. The analytical procedure has focused on the study of traces remaining of the procedure to prepare these materials (e.g., salts), and it has also studied the deterioration of the materials (e.g., proteins). Considering proteins, amide I, NH, and CH2 bands appeared with much broader bands indicating severe deteriorations. Several bands observed in model samples were also significantly decreased in intensity (e.g., phenylanine ring stretching band at 1004 cm−1), and the broad feature corresponding to the disulfide bridge (∼500 cm−1) disappeared. Raman spectroscopy was also applied to the study of hair proteins, notably keratin.120,121 In particular, FT Raman analyses of hair samples from a VIIth century cist burial121 have shown evidence for severe degradation and microbial colonisation. For example, the peak assigned to the disulfide bridge near 510 cm−1 is absent in the ancient sample (versus present in modern human hair; Figure 8). Another spectral signature of the loss of protein

generally they informed of the state of preservation of an ancient bone at the histological level. Besides, the bone preparation for SR FTIR imaging was also improved by embedding bone sections in a PMMA resin, which offers the possibility of studying brittle samples.101 This protocol was successfully applied to the study of fossil bone alterations. Recently, a study based on NIR-HCI combined with chemometric tools has evaluated the relative degree of the collagen preservation in bones from the Early Upper Palaeolithic, Mesolithic, and Neolithic periods.102 The discrimination of bones with/without collagen was performed by PLS-DA, and the evaluation of collagen preservation within the different strata was successfully achieved. Proteins were also probed using Raman spectroscopy both in paleontological and archeological samples.35,36 To minimize fluorescence emission background that covers the Raman signal (discussed in the previous section), a FT Raman device equipped with near-infrared laser excitation (e.g., 1064 nm laser) was mainly used. As described previously, the main features characterizing the proteins are the amide I band (∼1650 cm−1) and the amide III band (∼1245 cm−1); however, other features were also used as illustrated by the following examples. Applied to the study of ivory (generic term for exoskeletal tooth) that contains collagenous proteins embedded in a carbonated hydroxyapatite matrix, FT Raman was applied as an authentication technique of ivory and simulated ivory artifacts.103,104 In particular, the relative intensity ratio of the proteinaceous CH stretching band (∼2930 cm−1) over the hydroxyapatite PO stretching band (∼960 cm−1) was used as an analytical parameter. This intensity ratio parameter was used for analyses of ivories from several mammalian species and their discrimination.105,106 Raman spectroscopic characterizations of ivories from African and Asian elephants, woolly mammoths, sperm-whale, walrus, wart hog, narwhal, hippopotamus, and domestic pig were successfully shown. Chemometric analyses using PCA were implemented to FT Raman studies to improve the discrimination of the ivory from the different species.107 However, applied to the study of archeological ivories, degradation processes affecting organic components were pointed out, resulting in a difficult differentiation between mammalian species.108 For example, the study of an Early Bronze Age pig tusk did not show the amide I band at 1660 cm−1. Further analyses of ancient materials were proposed,109 and full vibrational assignments were done on organic (also inorganic) components of modern and ancient human teeth (IVth−XVIth centuries AD) showing specific vibrational features of Pro and Hyp in the 820−940 cm−1 region (∼10% of the total collagen). Modern and ancient teeth (150−6000 years before present) were also studied using Raman spectroscopy for dating experiments.110 Reliable results were obtained in the case of tooth enamel analysis (enamel is the most external part of the tooth). In particular, the intensity ratio of the 960 and 2941 cm−1 bands (PO and CH stretching bands) was correlated to the burial time (i.e., organic component intensity decreased in correlation to the duration of burial versus constant inorganic component intensity). However, no correlation could be obtained for the dentin (the dentin is covered by enamel in the tooth). The chemical changes in bone soft tissue related to burial were also studied111 on the basis of turkey bone fragments buried in soil until 64 days. Surprisingly (the diagenesis was previously associated with collagen loss), significant enhancements of the CH2 region (3040−2810 cm−1), amide I (1715− 1610 cm−1), NH (1500−1415 cm−1), and amide III (1358− 13


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Figure 8. Stack plot of (a) degraded hair fibers and (b) modern human hair. Reprinted with permission from ref 121. Copyright 2006 Springer.

Table 2. Fluorescence Emission of Media as a Function of Laser Excitation Wavelengtha excitation (nm) 248 collagen glues cross-linkage products amino acids and photooxidation products egg and casein amino acids and photooxidation products a

355

385 pentosidine 305 tyrosine (parchment)

440 pyridinoline 415 dityrosine, approximately 480 dihydroxyphenylalanine (DOPA), and related products

340 tryptophan; 420 dityrosine

435 ascribed to contributions from dityrosine (415) and N-formylkynurenine (NFK) (440), approximately 480 dihydroxyphenylalanine (DOPA), and related products

Reprinted with permission from ref 77. Copyright 2006 Elsevier.

structure is related to the α-helical amide I mode near 1650 cm−1 that is observed in the Raman spectrum of modern human hair. This band is absent in spectra of ancient fibers; it is substituted by a 1628 cm−1 band that informs of the presence of components arising from disordered and random coil conformations. A significant decrease in intensity was also observed for the amide III component near 1265 cm−1. Spectroscopic information from Raman spectroscopy provides a better knowledge of ancient samples; however, multidisciplinary approaches using complementary analytical techniques are nowadays preferred.122 2.2.2. Fluorescence Spectroscopy Applied to Paint Analysis. Fluorescence spectroscopy studies the emission wavelengths of molecules following their excitations by an irradiation source (e.g., ultraviolet lamp, laser). It represents a powerful analytical method for the noninvasive characterization of art materials such as pigments, dyes, and binders.123,124 In particular, proteins show an intrinsic fluorescence due to their aromatic amino acids (Phe, Tyr, and Trp). Several parameters may modify the fluorescence spectra such as protein structure or environment (examples given in the following paragraphs). Fluorescence spectroscopy allows the identification of fluo-

rescent degradation products such as pentosidine resulting from the Maillard reaction between proteins and saccharides.77,125 Laser-based techniques126 such as LIF were used in the early 2000s as a cleaning tool; for example, KrF excimer laser (248 nm) was used for the cleaning of tempera paints.127−129 In this context, the molecular changes of paint components induced by cleaning procedures were studied using several techniques such as LIBS (an atomic emission spectroscopy) and LIF.129 In particular, LIF spectra (KrF, 1.2 mJ cm−2 laser fluence) showed a broad feature at ∼450 nm for the unpigmented nonvarnished egg-based paint (paint layers ∼100 μm thickness) and extra bands corresponding to the pigments in the pigmented model paints. These features were used to probe the effect of the laserbased cleaning procedures depending on sample compositions (e.g., reduction of the LIF emission intensity for inorganic pigment sample areas). LIF was also applied to detect and discriminate proteins in several model binders (i.e., milk, egg white, egg yolk, and animal glues) prepared according to ancient recipes.77 On the basis of 1 mm2 samples, fluorescence emission spectra were recorded at two different excitation wavelengths, that is, 248 nm (KrF Excimer laser, 5 mJ cm−2 fluence) and 355 nm (third harmonic of a Q-switched 355 nm Nd:YAG, 5 mJ 14


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Figure 9. Fluorescence amplitude map and fluorescence lifetime map reconstructed for films of binding media egg white, egg yolk, and ox bone glue recorded with 337 nm excitation (the halo around the samples is ascribed to scattering from the background paper support; to the left of the samples, the fluorescence emission from adjacent disks is observed). Reprinted with permission from ref 132. Copyright 2007 Springer.

cm−2 fluence) (0.13 nm resolution obtained). The spectra showed differences related to amino acid compositions of the binders and to the presence of degradation products. For example, tryptophan-free collagen is discriminated from other tryptophan-containing proteins (such as egg and casein) and exhibited a strong emission at ∼340 nm. Another example is the detection of cross-linkage products; for example, collagen exhibits a ∼ 385 nm band related to pentosidine (mentioned previously). Table 2 shows markers that allow the egg proteins and casein to be differentiated from collagen.77 The interpretation of LIF fluorescence spectra is often complex (e.g., broad signals; difficult interpretation of spectra for mixtures); thus LIF experiments are combined with total emission fluorescence spectroscopy experiments130 that allow the acquisition of consecutive emission spectra recorded over a specific range (e.g., plotted spectra show fluorescence intensities in function of 220−400 nm excitation range and 250−600 nm emission range). Total emission spectroscopy provides additional information such as emission and excitation maxima of fluorophores and their wavelength dependencies, or information on the relationship between multiple fluorophores. The study performed on model egg-based media130 allowed precise fluorescence assignments (e.g., Trp, oxidation products of amino acids), permitting thus one to discriminate egg white and egg yolk. Additionally, the study showed that total emission spectroscopy can assist the choice of the laser wavelength for LIF spectroscopy. The changes in the fluorescence emission profiles due to the presence of pigments and to the exposure to light were also studied.82,131 The partial oxidation of Trp was shown, for example, in model casein-based binders following the light exposure; that is, the fluorophores observed at 360 nmexcitation/435 nm-emission were ascribed to N-formyl kynurenine and related fluorophores formed following the photooxidation of Trp. Total emission spectroscopy, LIF, time-resolved fluorescence spectroscopy, and FLIM124 were combined to discriminate binders such as egg-based media from glue.132 Time-resolved fluorescence spectroscopy is a wavelength-dependent graph of fluorescence lifetime (for specific excitation wavelength). For homogeneous materials, the same decay time is observed at any wavelength, while for a mixture of different substances, different fluorescence lifetimes should be observed. The FLIM technique maps the relative abundance of fluorescent materials in the

sample, and it reveals areas presenting different fluorescence lifetimes. In particular, FLIM showed differences in the kinetics of emissions of studied samples (films of egg white, egg yolk, and ox bone glue) (differences also observed using time-resolved spectra) (Figure 9). It allowed thus for one to discriminate among binding media via the maps of their spatial distribution. It can be pointed out that FLIM measurements provide quantitative measurements that are obtained using portable instruments.124 The SFS technique that represents an alternative and rapid method for scanning the fluorescence properties of a material has been also proposed for the differentiation of naturally aged protein-based binding media.133 Using the SFS technique, the spectrum of fluorescence emission intensity is recorded by varying both excitation and emission wavelengths simultaneously with a fixed offset (offsets of 30, 40, 50, and 60 nm between excitation and emission; excitation range: 220−600 nm; 1 nm resolution). To assess the differences in spectra of studied materials, a multivariate statistical analysis was implemented. Naturally aged binding media based on egg white, egg yolk, milk, casein, rabbit skin glue, ox bone glue, parchment glue, and fish glue were discriminated using SFS due to the presence of different groups of fluorophores in the different materials.133 In particular, spectral signatures (excitation wavelength associated with the peak in each of SF spectra) of Tyr, Trp, and various Maillard reaction products or amino acid oxidation products informed of the presence of a specific binder, for example, Trp (excitation wavelength ∼290 nm) in dairy proteins or eggs, and Tyr (excitation wavelength ∼279 nm) in collagen. The peaks associated with Maillard reaction products (excitation wavelength ∼345 nm) and amino acid oxidations (excitation wavelength ∼280 nm) were also considered to discriminate the binders.133 With an extended wavelength range (180−600 nm, i.e., VUV - visible wavelength range), the continuous tunability of the source and the brightness, the synchrotron VUV−UV−visible monochromatic beam was successfully evaluated on ancient samples (1 mm thick).134 The technique allowed acquisitions from a second to 1 min, and thus it has provided high-resolution spectra for the microspectroscopy and the full-field microscopy setups. The spectral resolution was 0.26 nm and the spatial resolution was 400 nm/590 nm for the microspectroscopy and full-field microimaging experiments, respectively. Applied to the study of a cello dating from the 15


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XVIIIth century, previously studied51 using FTIR (see related section), the spectra showed a typical emission profile of the glue.134 2.2.3. Electron Paramagnetic Resonance Applied To Study Protein Environment and Interactions. EPR (or ESR) is a spectroscopic technique that studies materials containing unpaired electrons such as paramagnetic species. Free radicals and some metal ions are typical paramagnetic centers. Contrary to NMR, it is electron spins that are excited instead of spins of atomic nuclei (the technique is based on a microwave irradiation inducing spin transitions of unpaired electrons in a sample located in a magnetic field). Samples can be analyzed in powder form (few milligrams) or in solid state for imaging experiments (area below 7 mm2). Applied to Cultural Heritage materials in the early 2000s, EPR was used (orthogonally to other analytical techniques) to probe the amounts of radicals during photochemical and thermal agings of triterpenoid varnishes.135 EPR was also used to study the complexes formed between copper ions and fatty acids or resins.136 Another works includes the EPR study of pigments such as Egyptian blue pigment (copper(II) paramagnetic species).137,138 Considering protein-based binders, EPR showed its capability to probe the protein environment using the copper(II) ion.139 The EPR signal of copper(II) is characterized by a quartet of lines at low field (2900−3300 G) and a strong line at high field (3300−3600 G). EPR spectra between 260 and 310 mT (T is tesla) have shown differences both in position and in shape for different binders and their environments. In particular, the data related to the hyperfine splitting of the quartet lines of the parallel region (Apar) and the g-factor of the quartet center (gpar) were used. Applied to the study of 3 mm2 samples of a 15th century manuscript, and in particular applied to one greenpainted sample, EPR spectra showed different values (Apar 170 × 10−4 cm−1, gpar 2.30) from those reported for Cu(II) present in paper (Cu(II) ion is often present in minor amounts in ancient papers (Apar = 192 × 10−4 cm−1, gpar = 2.25; and Apar = 129 × 10−4 cm−1, gpar = 2.31, respectively, attributed to copper bound to cellulose and copper bound to water)). These values were attributed to the coordination with nitrogen atoms, characteristic for copper−protein complexes. Using additional EPR experiments based on model paints with different copper-based pigments (verdigris, azurite, malachite) and binders (glue, egg white, egg yolk), verdigris and egg white were successfully identified as constituents of the studied ancient manuscript (Figure 10). Azurite and malachite showed different copper ion environments (attributed to the different basicities and solubilities of these pigments). The strong similarity of EPR spectra of the ancient sample versus egg white-based models suggests verdigris pigment bound to egg proteins. These conclusions were confirmed using complementary analytical techniques (IR, XRD, and XRF). Recently, EPR experiments were applied to the study of paint binders and their exposure to pollutants such as CO2, SO2, and NO2.140 EPR informed of the chemical reactions that may occur in a given environment. For example, EPR spectral features of a model sample containing an egg binder and exposed to NO2 (spectral weak line at higher g-factors) were attributed to a sulfonic free radical generated by the oxidation of the S−S cysteine bond of egg yolk. Thus, it showed strong reactivity of nitrogen oxide. EPR is commonly used in combinaison with other techniques that allow the identification of chemical products. These first studies dealing with EPR and protein-based

Figure 10. An EPR comparison of the copper parallel region for sample A and the reference samples. From top to bottom: azurite in yolk (NAY), malachite in yolk (NMY), verdigris in yolk (VY), sample A, verdigris in egg white (VW), verdigris in lapin glue (VL), malachite in lapin glue (NML), and azurite in lapin glue (NAL). Reprinted with permission from ref 139. Copyright 2013 John Wiley and Sons.

media are pointing out the highly informative capability of this technique, especially regarding the interactions between different paint components and their environment. 2.3. Time of Flight Secondary Ion Mass Spectrometry Imaging Applied to Ancient Samples

TOF-SIMS is a nondestructive technique (several types of measurements possible on a unique sample) allowing the simultaneous identification of both inorganic and organic compounds. The principle is based on the analysis of secondary ions emitted following a surface irradiation using primary ions beam. TOF-SIMS imaging has suffered until recently from the low efficiency of production of secondary molecular ions with gallium or indium LMIG.141 However, applied for the first time on a paint sample from The Descent from the Cross (Museo del Prado, Madrid) of the early Dutch painter Rogier van der Weyden (1399/1400−1464), this technique based on indium LMIG allowed for obtaining a map of the elemental distribution of pigments and molecular signatures of the oleaginous binding medium (whereas the presence of egg tempera was expected in the sample).142 The lateral resolution obtained was about 1 μm. The use of TOF-SIMS was highly improved by the development of cluster ion sources such as gold clusters (e.g., Au3+, Au9+, Au 400 4+ ) or bismuth clusters (Bi 3 + , Bi 5 2+ ). 141,143 As a consequence, the sensitivity, the mass range (until m/z 1500), and the lateral resolution were drastically improved.141 For example, bismuth clusters reached a lateral resolution below 400 nm.144 TOF-SIMS using a bismuth LMIG as the primary ion source was used to characterize ritual materials deposited on the surface of African wooden statuettes145 and patinas of African wooden statuette from the Dogon culture.146,147 In particular, protein fragments, lipids, polysaccharide fragments, urate salts, and minerals were identified, and the spatial distribution (i.e., elemental maps of each detected element on the analyzed area of the sample) was determined using TOF-SIMS experiments and confirmed with complementary analytical techniques (scanning electron microscopy with energy dispersive X-ray and FTIR microscopy). For example, blood was successfully identified in the patina from Dogon and Bamana sculptures via the characterization of heme (molecular weight 616.18 Da); heme is a complex between an iron ion and a protoporphyrin, and it is bound to hemoglobin.146 16


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TOF-SIMS analysis was also successfully applied to the demineralized tissue from Tyrannosaurus rex species (skull, vertebrae, femora, and tibiae). For example, the ratios of Gly and Ala fragments were used to inform of the presence of collagen α1 type 1 in the sample.148 Another study related to Equus evolution (using the genome sequence) used TOF-SIMS experiments in combinaison with other analytical techniques, to probe the presence of proteins in very ancient samples.149 In particular, horse bones recovered from permafrost (dated to approximately 560−780 thousand years BP) were studied, and TOF-SIMS revealed secondary ion signatures typical of collagen within the bone extra-cellular matrix; that is, TOF-SIMS spectra have revealed the presence of abundant secondary ions Gly, Pro, and Ala, whereas Thr, Gln, His, and Asp were detected at lower levels. More recently, a multilayered painting of Rembrandt was investigated with this technique showing unexpected materials.150 Pigments, binders, and various lead carboxylates resulting from interactions between lead white and linseed oil were identified (Figure 11). The study confirmed the presence of starch, which is surprising for Rembrandt paintings. Focusing on proteins, amino acid fragments were detected (Figure 11d) in the red lake (shearings of dyed cloth suggested by the study of historical written sources) but also within the cellulosic fiber, suggesting the presence of glue in the sizing of the canvas. This work demonstrated that TOF-SIMS imaging allows the accurate localization of the different compounds present in a sample and can be used as a screening method for the detection of proteins in Cultural Heritage samples. Recently, TOF-SIMS combined with PCA was evaluated to identify proteinaceous binders in several fresh samples and artificially aged model samples, as well as in samples dating from the XVth century.151 The experiments showed that the presence of inorganic compounds (e.g., calcium sulfate, pigments) modifies the mass spectrum of an organic binder (matrix effect). As a consequence, reliable discrimination between binders (such as casein and glue) remains difficult. Applied to the study of skin samples from a South-Andean mummy (XIth century),152 TOF-SIMS mapping was able to differentiate dermis and epidermis via differences in amino acid compositions of two major constitutive proteins, collagen and keratin. The epidermis composed of keratin was identified using Leu/Ile, Val, Phe, and Tyr fragments, whereas dermis, composed of collagen, showed the presence of Hyp (only present in collagen but not in keratin), Pro, Gly, Gln, and Ala fragments. The accurate localization of keratin and collagen in the studied sample was shown using, respectively, their Leu/Ile fragments and Hyp fragment. It can be pointed out that semiquantitative results obtained from Gly versus Ala intensity ratios (data based on the areas of the fragment peaks at m/z 30.03 and 44.05) for mummy skin dermis in this study152 were comparable to those obtained earlier on collagen of a Tyrannosaurus rex bone148 (values are, respectively, 2.1 versus 2.6). TOF-SIMS represents thus a powerful technique providing information on protein content, but also on the distribution of organic and mineral compounds in a single experiment and starting from a few sample amounts (e.g., investigated areas of 400 μm2 starting from a thin cross-section that is 0.5−1 μm thick).

Figure 11. High spatial resolution TOF-SIMS ion images: (a) Ion image of resin fragment ion (m/z 85, probably PMMA), corresponding to the embedding resin, which has gone into the sample cross-section. (b) Ion image of a polysaccharide fragment (C2H3O2−, m/z 59), corresponding to wheat starch in the second ground layer and linen on the canvas. (c) Ion image of the sum of fatty acid carboxylate ions, corresponding to lipids. (d) Ion image of the sum of amino acid fragment ions, corresponding to proteins on the canvas. (e) Ion image corresponding to the sum of lead positive ions (Pb+, Pb2+, and Pb3+). (f) Sum of ion images corresponding to lead white. Field of view 100 μm × 100 μm; 256 × 256 pixels; pixel size 390 nm. Color scale bars, with amplitude in number of counts, are indicated to the right of each ion image. The amplitude of the color scale corresponds to the maximum number of counts mc and could be read as [0, mc]. tc is the total number of counts recorded for the specified m/z (sum of counts in all of the pixels). Reprinted with permission from ref 150. Copyright 2011 American Chemical Society.

3. PROTEIN IDENTIFICATION BASED ON AMINO ACID COMPOSITION: APPLICATION TO CULTURAL HERITAGE SAMPLES 3.1. Chromatography-Based Techniques Applied to the Study of Amino Acids in Fossils

Since the mid 1950s, pioneer studies on fossils153−157 have investigated the possibility of identifying ancient proteins on the basis of their constitutive amino acids. The first evidence of the amino acid content was obtained from fossils (e.g., bones, shells, teeth) from different epochs and periods (e.g., Pleistocene epoch, 2 588 000 to 11 700 years ago; Pliocene epoch, 5.333 17


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composition of proteins from Pleistocene bones was Lys, His, Arg, Hyl, Asp, Hyp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, and Phe.158 Additional experiments based on electron microscopy have pointed out the presence of collagen and also the potential presence of other proteins besides collagen.158 Sample preparation for quantitative experiments was further optimized; in particular, a calcium salt removal step (that interferes with the ion-exchange column) was added, for example, precipitation using oxalic acid.161 Several other protocol optimizations were evaluated;165 for example, a fractionation using 10% CaCl2 (pH 9) following the HCldemineralization162 was tested to remove noncollagenous proteins (in the mid 1950s, the CaCl2-based procedure was described to separate mucoprotein and collagen in decalcified fresh bones,166 and it was used later in a modified version167 to study proteoglycan closely associated with mineral and collagen). Concerning bone demineralization, an EDTA-based treatment was also evaluated instead of a HCl-based treatment (10% EDTA, 7 days, 4 °C, pH 7.5, and presence of toluene to prevent growth of microorganisms).162 Neutral buffers were then used to purify the bone collagen in EDTA-demineralized residues (0.05 M tris-hydrochloride buffer or phosphate buffer with few drops of toluene, pH 7.5, 5 days, 4 °C). In particular, the procedure of demineralization with EDTA (with the additional use of neutral buffers) had resulted in a greater collagen extraction efficiency as compared to the one obtained using an acid solution followed by a CaCl2 treatment.162 On the basis of the optimized protocols, amino acid amounts of several fossils, including extinct species, were compared to recent ones. For example, amino acid contents of fossil bones dated from Pleitocene appeared similar to those of modern mammals; however, higher amounts of Ala and smaller amounts of Hyp were identified in the extinct ground sloth and the saber-tooth cat.162 These extinct species showed also a similar composition of amino acids to certain lower vertebrates (with larger amounts of Ala and reduced amounts of Hyp). However, the amino acid composition of the extinct western horse differed from the recent horse by its low His content.162 Applied to the study of protein residues from fossil oyster shells (dated from the Pleistocene to the Cretaceous, i.e., from 11 700 years ago to 145 million years), a similar content of amino acids was identified as compared to modern samples, with the difference that amino acid amounts declined severely from the Oligocene (33.9 to 23 million years).168 Older samples contained also less Asp and Gly.168 Analyses of older samples, for example, the dinosaur bones from Jurassic (201.3−145 million years) and Cretaceous (145−66 million years) periods, showed successful quantification of 19−20 amino acid residues.169 However, it can be pointed out that several unanswered questions and cautions were raised concerning the origin of identified amino acids (are these amino acids the surviving residues of proteins of original bones and tissues; are they formed during the decay of the animal carcass; or are they resulting from the recent activity of permeating soil microorganisms). The GC technique was also successfully applied to study the amino acids from fossils dated from the Pleistocene to Jurassic.170,171 Before analysis, the amino acid solution was cleaned with an ion-exchange resin column, and amino acids were converted into their volatile n-butyl-Ntrifluoroacetic acid derivatives. An internal standard (norleucine) was added to the analyzed sample. Applied to the study of fossil pecten shells, GC-FID had provided results similar to those of cation exchange chromatography but with 30−50 times less starting material.171

million to 2.58 million years ago; Miocene epoch, 23.03 million to 5.332 million years ago; Devonian period, 419.2 million to 358.9 million years ago).153−155,158 The classical sample preparation was composed of a cleaning step (e.g., sand removal, petroleum ether washing), a grinding of the sample (several grams to tens of grams), a fossil decalcification using trichloroacetic acid (2.5% TCA) or hydrochloric acid (1 N HCl), and a protein hydrolysis using concentrated HCl (6 N HCl, 100 °C, 24 h). The amino acids were then subjected to an analysis based on chromatography. In particular, two methodologies were first used (independently or jointly), paper chromatography and cation exchange chromatography. Paper chromatography is based on the separation of amino acids adsorbed on a cellulose paper using a solvent rising by capillary. Phenol was generally used as partitioning agent and ninhydrin as detection agent. Before paper chromatography analysis, the sample composed of amino acids was often desalted using cation exchange resin (amino acid elution with NH4OH).155 Analyses of ancient samples using paper chromatography resulted in the identification of several amino acids, for example, Ala, Glu, Gly, Ile, Leu, and Pro from Miocene shell (Figure 12),154 or Gly, Hyp, Pro, Ala, Leu, Glu, and Asp from Pleistocene bones.155,159 Complementary techniques such as electron microscopy155 or X-ray diffraction160 confirmed the presence of collagen.

Figure 12. Amino acids found in Miocene (25 000 000 years old) shell of Mercenaria mercenaria. The abbreviations used in the photograph are ALA, alanine; GLU, glutamic acid; GLY, glycine; ILEU, isoleucine; LEU, leucine; PRO, proline. Reprinted with permission from ref 154. Copyright 2006 John Wiley and Sons.

Cation exchange chromatography was used for quantitative studies.158,161,162 The analytical procedure163,164 was based on the measured absorbance of amino acids derivatized with ninhydrin after ion exchange separation. Quantitation was obtained by comparison with amino acid standards analyzed in the same conditions; results were finally expressed in micromole (or microgram) of amino acids per gram of sample. For example, results of three bones samples from Pleistocene were compared to a recent bone showing a 6-fold decrease in the amino acid content (e.g., total amino acid content of 31.748 μmol/g for a recent bone versus 5.012 μmol/g for an ancient one). However, ratios between individual amino acids were found to be similar in recent and old samples.158 It can be pointed out that several additional amino acids were identified as compared to paperbased technique results; that is, the identified amino acid 18


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fossils, effects of the sample environment (e.g., temperature; oxygen; surrounding sediments including the effect of soil microorganisms) on the organic matter and its degradation were considered and studied.154,156,168,177,178 For example, pioneering works in the mid 1960s on bones have suggested the potential presence of proteins other than collagen,158 and, on the other hand, some hallmark amino acids could not be detected in several study cases such as Hyp161 or Hyl for detection of collagens in fossils.179 Considering the environment of fossils, it was shown that several amino acids were identified both in the surrounding rocks and in the fossils.156 Thus, the interpretation of the amino acid-based analyses of the organic matter in fossils is complex, and the study of the organization and the survival of the organic compounds appears as critical.178,180 The deterioration of bones is described as resulting from several pathways of diagenesis.178 Focusing on the organic material, (i) chemical reactions may occur leading to changes in the collagen organization181 (potentially linked to the chemical deterioration of the mineral phase) and finally to gelatinization and collagen loss,178,182 and (ii) biodegradations may occur as a consequence of the microbiological environment.178,183 For example, fresh animal bones infected with soil microorganisms were studied and compared to archeological bones of known ages and burial conditions.184 In particular, the gel SDS-PAGE electrophoresis technique (used to separate proteins and degradation products according to their molecular weights through an acrylamide gel) showed that several extracted proteins from bones were broken into products of lower molecular weights in the case of unfavorable preservation conditions (e.g., sandy soil, seasonal floods). Supplementary experiments based on the cation exchange technique have shown a loss of Pro and Hyp. In addition, a preferential loss of amino acids with high numbers of carbon atoms (Tyr, His, Ile, Met) in poorly preserved samples was suggested.184 Another example showed similar chromatographic profiles (C18 column; proteins or amino acids derivatized with PITC monitored with UV detector at 214 nm) for HCl-based extracts of fossil bones and its sandstone matrix, suggesting the presence of same proteins in fossil vertebra of 150 million year old Seismosaurus dinosaur.172 Further analytical optimizations based on the use of a highly denaturant solution for protein extraction (guanidine HCl versus HCl) finally showed different HPLC profiles for fossil bones and its sandstone matrix. One potential explanation was that proteins may have formed complexes with minerals from bones,185 and complexes may have been broken in the presence of the denaturant solution. It can be pointed out that racemization was also used to study protein degradation186 and the intracrystalline organic fraction within biominerals.187,188 More broadly, it can be highlighted that racemization represents a dating technique189−194 because the biomolecular deterioration in the burial environment is slow. Racemization is associated with the slow conversion of the amino acids found in the L-configuration in living organisms to the D-enantiomers post-mortem, for example, L-isoleucine to Dalloisoleucine,189 L-aspartic acid to D-aspartic acid.195 Recently, experimental evidence for in-chain serine racemization was also provided on a model peptide (see details in section 5.4.1).196 Racemization is studied using liquid chromatography,188,194,195,197−199 gas chromatography,200 or, more recently, capillary electrophoresis-mass spectrometry (see details in the capillary electrophoresis section).201 The first LC strategy was based on cation exchange separation.197,202 Commonly, D and L isomers (1−20 μmol of individual amino acid) were derivatized

On the basis of these pioneer works related to the identification of the amino acid composition of ancient fossils and their quantitation, several objectives were investigated. The main topics that were explored were (i) better knowledge of protein contents including the study of the effect of surrounding sediments, (ii) better knowledge of the deterioration of the fossils organic matter (this issue is linked to the study of the organization of the fossil organic matter), (iii) dating experiments (because the molecular deterioration in the burial environment is slow, the organic matter represents an accessible tool for information), and (iv) better knowledge of the phylogenetic tree. It can be pointed out that all of these topics are linked. To unravel these issues, several improvements or modifications regarding analytical techniques or methods were proposed. Analyses of amino acids were often complemented with additional information on peptide/protein structures and their interactions (detailed in the following sections, in particular in the section related to immunological methods and the section related to proteomics). As described previously, the information on the amino acid content of fossil proteins was generally compared to the amino acid content of standard samples (i.e., comparison of the amino acids compositions and/or the individual amounts of amino acids of ancient samples versus recent samples), and it was often supplemented by complementary techniques (e.g., electron microscopy155 to observe collagen fibers). Thus, several hypotheses related to the amino acids-based signature of a given protein were proposed; for example, Gly, Pro, Hyp, and Hyl were often assigned to the presence of collagen.172,173 For example, the successful identification of Hyl and a high molar ratio of Gly suggesting the presence of collagen type I were shown on cancellous bone of Tyrannosaurus rex (late Cretaceous).173 Considering the technical viewpoint, a C18 reverse phase chromatography column was used for the amino acid identification (separation according to the hydrophobicity of amino acids). Amino acids were derivatized with FDAA and detected with a fluorescent detector at 340 nm. Additional experiments on the native extract analyzed by C18 high performance liquid chromatography (detection at 214 nm) have provided similar elution times for ancient samples and purified collagen type I and ostrich bone extracts. The source of the biomolecules identified was not considered in this study. However, on the basis of the complementary techniques used (HPLC, SEM/TEM, confocal microscopy, and electron diffraction analysis), hypotheses on minimal signs of permineralization of bony tissues of Tyrannosaurus rex were provided, and thus on the lack of extreme diagenetic alteration that promotes the preservation of ancient biomolecules.173 Another case study showed that high amounts of Asp, Gy, and Ser were found in an 80 million year old mollusk shell.174 The molar amounts released assumed the presence of a repeating protein sequence (-Asp-Y-)n where Y was Gly or Ser,175 similarly in ancient and recent samples (e.g., Asp:Gly:Ser 6.3:1.7:1 in Scabrotrigonia thoracica fossil and 7.2:1.7:1 in Neotrigonia margaritacea extant). However, cautions against the use of total amino acid compositions were provided.176 In particular, experimental data obtained from the analysis of an organic matrix of coretop and fossil planktonic foraminifera (2000−4000 BP to 300 000 BP) have suggested that hydrophobic polypeptides may be preserved in much older specimens but some of the acidic polypeptides are lost, and the presence of adsorbed/contaminant components cannot be ruled out.176 It can be pointed out that since the beginning of the studies of amino acids from 19


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of ninhydrin derivatives was proposed in 1969.223 As described in the previous section, the amino acids resulting from hydrolyzed proteins (approximately 4 mg of starting material) are loaded onto a sulfonic acid resin column at pH 3. They are then gradually eluted by a pH and salt gradient. The amino acids are eluted in a specific order related to their isoelectric points and their charges, and they can be detected with a good accuracy (∼3%) by ninhydrin detection (in optical density). Norleucine is added in experiments as internal standard for quantification. Because of its accuracy and by the use of an internal standard, the method succeeded in the identification of gelatin, casein, and egg (white and yolk), all used as binders, by their distinctive amino acid compositions. Gelatin could be distinguished from other binders by its Hyp content greater than 2% (and for confirmation: more than 25% Gly, over 10% Pro and Ala, less than 5% Val, Leu and Lys, less than 3% Ile). Casein was characterized by a Glu percentage greater than 15% (and for confirmation: less than 8% Asp, less than 7% Ala, and more than 8% Pro). Also, egg white and egg yolk have more than 8% Asp and can be discriminated on the basis of their Ser and Thr contents (3−4% versus 5−8% for Thr and 7−8% versus 9−12% for Ser, respectively, for egg white and egg yolk). This method was successfully evaluated on ancient samples. Gelatin was, for example, characterized in a parchment sample from the XVIth century.223 Reverse phase high performance liquid chromatography (C18 stationary phase) coupled to UV−visible spectrophotometry was also used in the early 1990s for the analysis of amino acids in artwork samples.224−226 To improve the sensitivity of the technique, the amino acids were derivatized with PITC (also called Edman’s reagent) to form phenylthiocarbamide (PTC) amino acid derivatives. The derivatization procedure was based on the semiautomated workstation (PicoTag) that prevents contamination and allows better reproducibility. Norleucine was also used as internal reagent for quantitation purpose. The technique was used for the identification of proteinaceous binders from the panels “The Annunciation with Saint Francis and Saint Louis of Toulouse” painted by Cosimo Tura (1475).224 On the basis of hydrolysis with HCl (6 N), the obtained chromatograms revealed amino acid profiles of egg yolk for red and brown pigmented areas, while animal glue was characterized for blue-pigmented areas. A medium mixing the glue and the egg yolk was also suggested for the green area. Besides, the analysis showed some interferences with particular pigments (e.g., copper carbonate, calcium carbonate) resulting in a loss of peak intensities for several amino acids that affect the binder identification. An alternative method based on extraction using NaOH (1 N) was proposed to identify protein binders in various artwork samples including polychrome stone sculptures dating from the XIIth−XIIIth centuries, the gesso ground of a wooden statue dating from the XVth century, plasters dating from XVIth century, and frescoes and stuccoes from various ages from a cathedral of the XIIth− XIIIth centuries.226 Before the quantitative analysis using C18HPLC, the hydrolyzed sample was desalted using a sulfonic resin. Among the results, an amino acid composition similar to egg yolk was observed for the polychrome sculpture samples, whereas the detection of Hyp and Hyl allowed the identification of gelatin in the ground of the gesso from the wooden statue, which agrees with ancient recipes. Other amino acid derivatization agents for artwork analysis are also described such as FMOC-Cl,225 OPA/2-ME,227 or AccQ fluorescent reagents.228 To obtain higher confidence levels for binders identification, statistical treatments of the data were proposed

using an L-amino acid N-carboxyanhydride (NCA) to obtain diastereoisomeric dipeptides;197 that is, L-leucine-NCA or Lglutamic acid-NCA was generally used (L-glutamic acid-NCA was preferentially used with phenylalanine, tryptophan, and basic amino acids).195,197 The relative chromatographic positions of diastereoisomers were different according to pK values.197,203,204 Reverse phase chromatography (coupled to fluorescence detector) was also proposed to detect amino acids in the subpicomole range.198 The method is based on derivatization of amino acids with o-phthaldialdehyde with the chiral thiol, N-isobutyryl-L-cysteine, to produce fluorescent diastereomeric derivatives of chiral primary amino acids.196,198 Racemization can be applied to various types of fossil samples and environments192 allowing the study of several key questions among which are dating192 but also geochronology,194 diagenesis,186,205,206 and biomineralization or taxonomy studies.207 For example, concerning biomineralization, a recent study showed that compositions of intracrystalline proteins from shells of marine gastropods (Patella vulgata) are dominated by acidic residues, which are resistant to bleaching probably because of their strong interactions with the mineral phase.205 Racemization shows several main strengths; however, its major drawbacks are related to its sensitivity to the sample environment (such as temperature, leaching effect, pH, etc.)186,193,200,206,208,209 and to structural modifications of proteins; these drawbacks may impact the accuracy of results. In particular, comparing the dating results of racemization and other techniques such as radiocarbon analysis (the decrease of 14C radioactive isotope210−212 used for post-mortem dating) remains difficult.213−216 Thus, studies and developments are constantly proposed for a better knowledge of racemization limits and its reliability.217−219 For example, differences in patterns of diagenesis at high and low temperatures (well-dated corals grown in isothermal conditions as a “natural” diagenesis experiment) were used to develop a new mathematical approach for the calculation of the relative reaction rates and the temperature sensitivity of major degradation reactions.219 3.2. Analysis of Amino Acids from Artwork Using Paper Chromatography and Liquid Chromatography

The first studies of the amino acid composition of artwork binders were provided at the end of the 1950s using paper chromatography (technique detailed in the previous section) on several canvas and mural paintings from Roman, Chinese, and ancient egyptian materials from different periods (XIIIth to XVIIIth century).220 Samples were hydrolyzed using a HClbased solution, and a cellulose-based Whatman paper was used as separation support. Butanol mixed with acetic acid and water (60/15/25) was used as mobile phase. The main objectives were (i) to show the presence or absence of amino acids, and (ii) to differentiate the binders based on egg and glue, the latter being characterized using the presence of Pro and Hyp on the chromatogram. Staining was performed using ninhydrin (resulting in yellow spots on white background)38,220 or isatin (resulting in deep blue spots on orange background).220 Dansyl amino acid derivatives221 were also proposed to improve the detection sensitivity by their ultraviolet fluorescence below 10−10 mole. The experiments were successfully performed on various paper-chromatography supports such as silica, alumina, or polyamide-cellulose spread on a glass slide.221,222 Because this technique was not resolutive enough nor discriminative or quantitative for binder identification, a method based on ion exchange chromatography164 and optical density measurement 20


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Figure 13. UPLC chromatograms obtained from model samples: (a) egg tempera; (b) milk tempera; and (c) glue tempera. Reprinted with permission from ref 231. Copyright 2012 Springer.

using two-dimensional ratio plots, SCF, and SIMCA.229 Several technological improvements were also evaluated as the use of columns with lower internal diameters (microbore column with 2.1 mm inner diameter instead of classical 4.6 mm inner diameter; and reduced mobile phase flow of 0.5 mL/min instead 0.8−1.5 mL/min) allowing results similar to the traditional method in terms of efficiency, runtime, LOD, but with a lower mobile phase consumption.230 The breakthrough in terms of sample consumption, total time analysis, and reproducibility from experiment-to-experiment231 was provided by the use of UPLC equipped with columns packed with particles of 1.7 μm size.232 Used in a combined analytical approach with immunological analysis, targeting accurate egg proteins such as ovalbumin (see details in the immunological methods),231 the C18-UPLC technique showed its abilities to provide resolutive

separation of amino acids from various tempera samples in 8 min total run time231 (Figure 13) (instead of 15−40 min using classical HPLC equipement), starting from a few pmol/μL of standard amino acid samples (where classical HPLC claims 10− 100 pmol as detection limit228 depending on the derivatization method used and the chromatogaphic parameters). In this study,231 norvaline was used as internal standard, and amino acids profiles from egg-based binders were compared to those of milk, glue, and egg−glue binders. Whereas the studies of single binders were successful, it was shown that the detection of amino acids was disturbed in the case of the binder mixture of different origins in the presence of particular pigments. For example, glue was not detected from an application of a glue red ochre layer to a pre-existing egg-based layer. 21


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Another technical improvement applied to the study of modern mural paintings was realized using an HPLC separation method coupled to MS using a derivatization-free methodology.233 Following acidic hydrolysis of proteins with HCl, underivatized amino acids were separated using a hydrophilic interaction chromatography column (ZIC − HILIC; a zwitterionic stationary phase covalently attached to porous silica). In this study, a ZIC − HILIC precolumn (2.1 mm × 20 mm; protective role of the analytical column) together with a ZIC − HILIC analytical column (2.1 mm × 150 mm) were used. The MS data were collected in MRM mode, which allows the selective monitoring of ions of interest and their fragments; that is, each amino acid is characterized by specific pair formed by parent ion and fragment (daughter) ions as shown in Table 3. The MRM method allows a selective quantification of a target compound in very complex mixtures up to 6 orders of linear dynamic range, reaching a high LOQ (lowest concentration where the relative uncertainty on a single measurement is reproducible within ±20%); for example, in this study,233 the LOQ of Pro was 0.08 pg/μL (i.e., 695 amol/μL). Quantification was obtained using 13C-labeled amino acids as internal standard. Different amino acid profiles were produced for egg, casein, or glue binders mixed with an inorganic pigment (titanium dioxide). On the basis of amino acid concentrations and the comparison to model paints, the study of modern tempera paintings showed successful characterization of the glue binder in the DeLuigi’s paintings233 due to high concentrations of Pro, 4-Hyp, Glu, and Asp (and lesser concentrations of Ala and Leu). Both egg and casein binders were characterized using the prevalence of Leu, Ile, and Asp for the Sironi’s painting.233 An alternative protocol, allowing the analysis of underivatized amino acids, was also described.234 The separation was performed on a C18 column, and MS detection was performed in MRM mode but without IPA that is usually required for the separation of underivatized amino acids using reversed phase liquid chromatography (e.g., trifluoroacetic acid). The analysis of samples of mural paintings from the Ethiopian UNESCO-listed church of Yemrehanna Krestos dating from the XIIIth century showed the presence of casein and egg clusters.234 The method was also employed for the study of microsamples of 19 paint layers and 11 ground layers of Jacek Malczewski’s paintings dating from the XVIIIth and XIXth centuries, revealing that the artist used proteinaceous binders (glue, egg, or casein) only for the ground preparation.234

Table 3. Transitions Monitored and the Compound Parameters DP, CE, and CXP Optimized for the Analytesa Q1 m/z

Q3 m/z

DP (V)

CE (V)

CXP (V)

[Leu + H]+

132.10

[Ile + H]+

132.10

[Phe + H]+

166.10

[13C3Phe + H]+

167.00

[13C1Leu + H]+

132.90

[Thr + H]+

120.10

[Met + H]+

150.20

[Val + H]+

118.10

[13C1Val + H]+

119.00

[Pro + H]+ [13C1Pro + H]+

116.00 117.20

[3−4-Hyp + H]+

132.00

[Tyr + H]+

182.10

[Ala + H]+ [13C3Ala + H]+ [Glu + H]+

90.00 93.00 148.20

[13C5Glu + H]+

153.00

[Ser + H]+

106.00

[cysteine+ H]+

241.10

[Asp + H]+

134.00

[13C4Asp + H]+

138.00

[Arg + H]+

175.10

[13C6Arg + H]+

180.80

[Lys + H]+

147.20

[His + H]+

156.00

86.10 69.20 86.10 69.20 120.10 102.90 121.10 104.00 86.10 44.10 102.90 77.00 133.00 104.00 72.10 55.10 72.00 55.00 70.10 70.20 68.10 91.00 86.10 68.10 165.10 136.00 91.20 44.20 46.20 129.90 102.10 84.10 134.90 106.00 88.20 88.00 60.20 152.10 120.10 73.90 88.10 73.90 91.10 75.90 116.00 70.20 135.00 121.10 74.00 130.00 84.10 110.10 83.20

41.00 41.00 41.00 41.00 43.00 43.00 51.00 51.00 45.00 45.00 78.00 78.00 39.00 39.00 35.00 35.00 45.00 45.00 42.00 40.00 40.00 49.00 49.00 47.00 43.00 43.00 43.00 32.00 37.30 40.00 40.00 40.00 39.00 39.00 39.00 34.00 34.00 51.00 51.00 51.00 34.00 34.00 42.00 42.00 53.00 53.00 55.00 55.00 55.00 32.00 32.00 46.00 46.00

15.26 25.60 15.26 25.60 18.30 37.90 18.90 37.80 16.40 31.00 25.70 36.00 14.50 15.30 16.80 30.00 17.80 29.00 23.80 24.30 24.30 19.20 21.40 30.80 15.00 20.70 40.00 17.20 18.70 14.05 16.60 23.80 13.30 18.90 24.10 14.60 16.60 20.18 27.90 41.80 14.70 20.80 14.90 21.90 20.90 33.00 22.06 20.90 31.00 14.80 23.60 20.20 35.60

15.70 12.50 15.70 12.50 21.80 19.00 11.00 9.50 6.50 7.70 18.20 14.00 23.10 18.50 12.80 9.50 6.00 5.00 12.80 6.00 6.00 7.50 7.20 6.00 15.20 12.40 8.30 7.20 7.50 23.10 18.30 15.50 12.00 9.50 7.80 15.50 10.30 14.00 11.00 13.60 15.70 13.20 9.50 6.50 21.00 12.70 12.50 11.40 6.50 23.10 15.20 19.80 14.60

precursor ion

3.3. Analysis of Amino Acids from Artwork Using Gas Chromatography-Based Techniques

3.3.1. Gas Chromatography Applied to Ancient Samples. GC analysis still remains one of the most used techniques to characterize binders in artwork samples.235−237 As introduced in a previous section of this Review (related to fossil analysis using chromatography), the methodology is based on the determination of the amino acid content separated by a fused-silica capillary column. A preliminary hydrolysis step is required to transform proteins into their amino acids (HCl commonly used), and the subsequent derivatization procedure aims to modify the amino acids into volatile compounds suitable for GC analysis. One of the first publications referring to the GC technique for the analysis of artwork is dated from the late 1960s.38 In this work, GC was used to characterize different types of binders, adhesives, or varnish, in a way complementary to other analytical techniques such as paper chromatography. Focusing on proteins, amino acids were obtained using a sulfuric

a

Symbols used: Precursor ion selected in Q1 (Q1), fragment ion selected in Q3 (Q3), declustering potential (DP), collision energy (CE), collision cell exit potential (CXP). Reprinted with permission from ref 233. Copyright 2010 Elsevier.

22


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Figure 14. GC/MS profile of the amino acid and fatty acid derivatives of the paint sample B17, obtained by the scan mode (a) and by the SIM mode (b). Experimental conditions and abbreviations are given in the text (see related publication). Reprinted with permission from ref 246. Copyright 1995 Springer.

terms of sample preparations. In particular, a purification step combined with protein hydrolysis (10 μg of starting proteinaceous material) using cation exchange238,239 was proposed to reduce the loss of amino acids resulting from the formation of Maillard products (condensation reactions between aldehyde functions of polysaccharides present in the sample and amine groups of amino acids).240 The hydrolysis/cleanup procedure carried out under mild conditions was based on the catalytic cleavage of the amide bond of the amino acid chain of proteins by H+ ions adsorbed on a strongly acidic cation exchanger (110 °C during 24 h). With this method, amino acids were immobilized on the resin while carbohydrates were eluted, which prevents the formation of condensation products.239 The amino acids eluted from the cation exchanger (with 7 M ammonia) were then submitted to a two-step derivatization procedure: acidic functions were esterified with butanol, and amine groups were acylated using TFAA. This procedure showed also its efficiency against interferences that may be caused by pigments in studied samples. While the classical methodology (hydrolysis using HCl without ion exchange cleanup) showed large fluctuations of relative concentrations of several amino acids (e.g., Ser, Thr) in samples containing calcium-based or iron-based pigments (e.g., chalk CaCO3, yellow ochre Fe2O3·H2O) (that may result in an incorrect identification

acid-based hydrolysis of the proteins (cleavage of amide bonds of proteins), and trimethylsilyl amino acid derivatives (amino acid derivatization with bis(trimethylsilyl)acetamide) were analyzed using GC equipped with a FID detector (consists of the detection of ions produced during the combustion of organic compounds in a hydrogen flame). However, an instability of amino acid derivatives was observed, and difficult interpretations of data obtained for ancient binders were noticed. Another amino acid derivatization method was introduced in the mid1980s for artwork studies.238 The amino acids resulting from 6 M HCl hydrolysis were derivatized into their N-acetyl methyl esters.238 For example, animal collagen could be differentiated from other proteinaceous binders by the presence of Hyp and significant amounts of Gly and Pro. To discriminate casein from egg white and egg yolk, the following amino acid ratios were used (evaluation on model samples): Leu/Ala; Pro/Ala; Pro/ Ser; Pro/Leu; Glu/Asp (e.g., the Glu/Asp ratio is of the order 2/ 1 for casein, while it is 1/1 for egg). This methodology was successfully applied to paint and ground samples from various artwork. For example, Hyp was identified in sample of gesso from “The Madonna of the Meadow” painted by Giovanni Bellini (XVth century), indicating that collagen was used for the gesso preparation.238 Following these first applications of GCFID to artwork samples, several improvements were proposed in 23


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Figure 15. PCA score plot of amino acid relative percentage contents of reference samples (○) and of old wall painting samples (●) V11, V9, Z3 from Giudizio Universale and AFF from Pisa Monumental Cemetery, obtained by suppressing pigment interference following the ammonia extraction method (N) or the cation-exchange resin method (D). Reprinted with permission from ref 259. Copyright 1999 Elsevier.

of the binder), the procedure based on a preparatory step using ion-exchanger did not suffer from interferences.239 An alternative GC-FID procedure based on the use of ECF derivatization reagent to transform amino acids into their ester derivatives (i.e., N(O,S)-ethoxycarbonylamino acid ethyl esters)241 was proposed for the study of the binders from Cultural Heritage works.242−244 This procedure allows a fast derivatization reaction, that is, a few minutes versus several hours to 24 h for other derivatization procedures.243 The other advantage of this derivatization method is the simultaneous analysis of amino acids and fatty acids,242,244 particularly interesting for samples containing mixtures of lipids and proteins. On the basis of the ECF derivatization technique coupled to GC-FID analysis, the effect of pigments and aging on amino acid composition was pointed out;245 in particular, the detected concentrations of Asp, Glu, Ser, and Thr were affected by the presence of hematite, ochres, umber, azurite, and malachite. However, the concentrations of amino acids substituted with alkyl groups (Ala, Val, Ile, Leu, Gly) and imino groups (Pro, Hyp) were found to be less affected by pigment interferences. Aging also showed effects on the total protein content of several binders such as egg yolk.245 Besides the improvements related to the derivatization methods, instrumentation was enhanced in the late 1980s− early 1990s by the introduction of MS for the identification of protein binders in artwork (starting from less than 1 mg sample amount), that is, GC−MS246 and pyrolysis GC−MS247 (pyrolysis GC−MS is detailed in the following paragraph). The coupling of GC with MS makes this technique more informative than GC-FID due to the accurate information obtained on the MW of amino acids. The information related to MW is also often coupled with the information related to GC retention times for a greater confidence in identification. MS provides also structural information on analyzed compounds, which makes possible the identification of new molecules in a sample such as unpredicted compounds (binders other than those commonly used). Concerning MS-based quantification,

several methodologies using various instrument geometries can be applied or used as described below (e.g., using SIM mode analysis246,248) and in the following section related to mass spectrometry (see the section dedicated to proteomics for details). By combining the separative capability of GC and the specificity and measurement accuracy of MS, it is thus possible with GC−MS to study complex mixtures made with proteinaceaous media and other ones such as lipids in a single analysis run.246 Applied to the study of medieval painting media,246 the first procedure described was based on HCl hydrolysis (5−20 mg starting material; norleucine as internal standard), with or without cation exchange purification (due to the loss of fatty acids), and derivatization of amino acids into their N-trifluoroacetyl-O-2-propyl esters, and fatty acids into their 2-propyl esters. The GC−MS instrument used in this study246 was equipped with capillary columns of low polarity (e.g., methyl silicone type), an EI ionization source (radical cation and its possible fragmentation produced using highly energetic electrons), and a quadrupole analyzer (ions separated using the stability of their trajectories in the oscillating electric fields applied to rods by application of radiofrequency and direct current). This analyzer had a dynamic range of about 3−4 orders of magnitude (currently the quadrupole has 5−6 orders of magnitude) particularly interesting for quantitative analysis. In particular, MS detection was operated using SIM; that is, only the mass-to-charge ions of interest (i.e., characteristic of a particular binder) are detected resulting in more informative spectra as shown in Figure 14.246 The methodology allowed the detection of 10 pg of target compounds. Various proteinaceous binder models were characterized by their relative amounts in amino acids, and the data were processed using PCA diagrams to facilitate the recognition of protein sources. Applied to ancient samples, the methodology identified high contents of Gly and Pro in the paintings of the Baptistery dome in Parma (Italy, executed by Byzantine masters around 1250), indicating that animal glue was used as binder alone or in a mixture with other binders (depending on the localization of analyzed samples) 24


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hydrolysis (MTBSTFA for the derivatization of amino acids). The use of chelating agents such as Na2EDTA250 was also proposed to suppress the pigment interference via the formation of complexes with metallic cations. The method is based on the formation of metallic ion−EDTA complexes following the hydrolysis step and before the amino acid derivatization (using ethyl chloroformate). The method was successfully applied to samples of wall painting from the “Fumoir” room in the palace of the Marqués de Montortal (XVIIIth century, Carcaixent, Spain). In particular, PCA plots obtained from the statistical treatment of GC-based data showed that the samples treated with EDTA were closer to the gelatin cluster than those without the EDTAcomplexation method. The EDTA-cleaning procedure allowed thus a better discrimination of protein binder for a more consistent identification.250 It can be noted that this cleaning procedure was also proposed for HPLC-based analysis.262 The use of barium chloride (following the hydrolysis of the sample and before the amino acid derivatization) was also proposed to suppress interferences due to sulfates.263 As previously introduced, the GC-based analysis allows the identification of proteinaceous binder(s) (commonly egg, milk, and/or glues) by comparison of amino acid contents of studied samples versus reference samples (often with the help of statistical treatments).237 The technique can be applied to various types of samples such as wall paintings,264−266 and frescoes,267 canvas paintings,268 or paintings on wood panels,269 polychrome and gilded decorations of walls,270 adhesives in the wooden backing of paints,271 binding media in icons,272,273 or polychrome sculptures,248 ancient Egyptian cartonnage with polychrome decoration,274 and ancient musical instruments.275 The main difficulties of the technique remain (i) the presence of complex binder mixtures from original artwork or from restoration works,271 (ii) the presence of unexpected proteins, voluntarily added in artwork by artists or resulting from contaminants (e.g., fungi),236 and (iii) the case study of highly degraded samples due to aging, UV light (e.g., slight decrease of relative amounts of Tyr and Lys was observed by GC−MS using sample exposures at 254 and 366 nm),276 or due to the presence of neighboring components, pollutants, etc. These case studies show generally several changes in the resulting amino acidic profiles. Consequently, the preliminary study of adapted models (e.g., mixtures of animal glue and garlic)277 or the use or complementary techniques to confirm the results (see details in sections related to proteomics or immunological techniques) are often required. Racemimization was also used to study mixtures of proteinaceous adhesives (glue and casein).271 The hypothesis provided was the induced racemization of L-Asp to D-Asp of animal-skin glue during its preparation by strong heating. It can be pointed out that GC−MS remains a main technique for binder identification, but it is nowadays mainly used in combination with complementary analytical techniques to certify results, to obtain a complete characterization of the studied artwork (organic and inorganic media), and/or to evaluate their state of conservation/degradation at the molecular level; for example, among the complementary techniques used are found synchrotron radiation278 micro-Raman279 and/or micro-FTIR spectroscopy,280,281 optical and/or electronic microscopies,282,283 X-ray-based techniques,284 and more recently tandem mass spectrometry (see proteomics section).285−288 3.3.2. Pyrolysis Gas Chromatography and Pyrolysis Gas Chromatography−Mass Spectrometry Applied to Ancient Samples. The application of pyrolysis GC (commonly

such as casein/milk that was detected with its high amount of Glu and the presence of myristic acid.246 Various modifications were suggested to improve the GC− MS procedure. They mainly refer to sample preparation, that is, purification and/or amino acid derivatization.235,237 Considering derivatization, three main procedures can be pointed out for their improved applications to the analysis of Cultural Heritage artwork: (i) derivatization with alkyl chloroformates such as the previously described ethylchloroformate-based derivatization to obtain N(O,S)-ethoxycarbonylamino acid ethyl esters;241,249,250 and more recently methylchloroformate;228 (ii) the previously described derivatizations with trifluoroacetamide and methanol,238 butanol,239 propanol,246,251,252 and, more recently, with pentafluoropropionic anhydride253 to obtain respectively Ntrifluoroacetyl methyl esters, butyl esters, propyl esters, or Npentafluoropropionyl n-propyl esters (reactions in two steps: esterification of the acidic functions with alcohol, then trifluoroacylation or pentafluoropropionylation of the amine group); and (iii) derivatization with alkylsilyl acetamide as introduced previously with N,O-bis(trimethylsilyl)acetamide38,254 and more recently MTBSTFA255−258 to obtain respectively trimethylsilyl derivatives and tert-butyldimethylsilyl derivatives. Using this last method, it was proposed to use microwave-assisted hydrolysis (6 M HC1) prior to the amino acid derivatization with MTBSTFA to reduce the hydrolysis time to 55 min (up to 24 h for common procedures).255,256 Different sample preparation procedures were also investigated to reduce the inferences caused by the presence of pigments. For example, a study compared two different cleanup procedures to suppress pigment interferences.259 The first one consisted of extracting the proteins from the paint sample with a solution based on ammonia prior to the hydrolysis step (2.5 M ammonia, 3 h, 60 °C, ultrasonic bath). The second one was based on a cation-exchange cleanup of the acid hydrolysate. The technique based on cation exchange also allows inorganic salts removal. Among both cleanup techniques, ammonia extraction prior to the hydrolysis step provided a better recovery of proteins (except in the presence of copper ions). Combined with statistical treatment by PCA of the relative percentage of amino acids for each studied binder (providing clusters containing the same binder), both procedures were successfully applied to samples of ancient wall paintings, that is, three samples from the ‘“Giudizio Universale”’ in Florence Cathedral and one sample from the back of the frescoes of the Monumental Cemetery in Pisa. While no reliable binder identification was obtained for these samples when they were analyzed without cleanup procedures, using the cleanup methods, the PCA data obtained for all of the old samples were very close to the milk binder cluster (Figure 15).259 Alternative procedures to eliminate pigments or organic components were based on reverse phase purification (C4 or C18) after ammonia-driven protein extraction. 260,261 In particular, the C4 separation was added to a multistep chemical pretreatment including several extractions, derivatizations, and hydrolysis for the combined study of proteinaceous binders, glycerolipids, natural waxes, terpenoid resins, and polysaccharide media, originating from the same paint sample.261 Focusing on proteins, the soluble fraction obtained by ammonia extraction (2.5 M) (in ultrasonic bath) was dried and reconstituted in trifluoroacetic solution (1%). A further diethyl ether partitioning step was performed to remove remaining lipid-resinous materials. The C4 purification was subsequently used to separate saccharide and protein fractions before their dedicated 25


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Figure 16. Pyrogram of sample 1 (see related publication for details). Identified compounds: (1) pyrrole; (2) toluene; (3) 2-methylpyrrole; (4) 3methylpyrrole; (5) ethylbenzene; (6) styrene; (7) C2-pyrroles; (8) benzonitrile; (9) cresol; (10) phenylacetonitrile; (11) phenylpropanenitrile; (12) indole; (13,14) n-1-alkenes; (15) dibenzyl; (16) diketodipyrrole; (17) diketopiperazin. Reprinted with permission from ref 317. Copyright 2006 Reed Elsevier Inc.

equipped with hydrogen flame ionization detector) to the study of ancient samples was investigated since the mid 1960s;289 it was mainly applied to the study of drying oils and resins.290 The coupling of pyrolysis GC with MS allowed the technique to reach its full potential291 in particular for the studies of different classes of molecules (such as lipids, carbohydrates, proteins) in artwork247,292−296 or archeological materials.290,297,298 From a technical point of view, samples (tens to hundreds micrograms) are placed in a quartz tube or in contact with a platinum wire and are thus heated to high temperatures (500−1000 °C temperature range). Three types of pyrolyzers are used,295,296 the microfurnace (pyrolysis chamber made with steel tube; the temperature is raised to the pyrolysis temperature and maintained for a selected duration), inductive heating/Curie point (based on ferromagnetic conductor heated by interaction with radiofrequency; the temperature is limited to the materialspecific Curie point temperature for which transition from ferromagnetic to paramagnetic properties occurs), and resistive heating using a metallic filaments (platinum resistance coil). Concerning the MS equipement, EI (detailed in the previous section) or CI (ions produced through collisions of the analyte with ions of reagent gas) are the main ionization sources used. Also, quadrupoles (detailed in previous section) or ion traps (3D quadrupole based on static direct current and radiofrequency oscillating electric fields to trap ions) are the most commonly used MS analyzers. The thermal decomposition of the sample generates pyrolysis products that are separated by GC and detected by MS. To obtain a better chromatographic separation, a derivatization step of analytes is generally added.295,296 One of the most used derivatization procedures is THM.299,300 It is based on the sample pyrolysis in the presence of the TMAH methylating reagent that results in the sample hydrolysis and the subsequent alkylation of esters and phenolic compounds.299 The method showed its capability for the study of several types of compounds such as resinous ones, fatty acid triglycerides, natural waxes,299 but also amino acids.301,302 To reduce side reactions mainly relating to fatty acids (e.g., the isomerization of unsaturated fatty acids or α-methylation of acid moieties),303 alternative derivatization procedures were proposed with the use

of trimethylsulfonium hydroxide304 or tetramethylammonium acetate.305 Silyl-based derivatization agents were also proposed such as N,O-bis(trimethylsilyl)trifluoroacetamide 306 or HMDS.307 Applied to the study of proteins from ancient samples, TMAH308 and HMDS309−311 derivatizations were mainly used. The study of the pyrolysis products of target amino acids (e.g., Val, Leu, polyglycine, polyalanine) has revealed the complexity of the analytical approach.312,313 In particular, among the products of the pyrolysis, several carboxylic acids, primary and secondary amide compounds, as well as several components resulting from amino acid intermolecular condensation were identified. However, studies of reference binders (i.e., milk casein, egg yolk, egg albumin, bone glue, skin glue, rabbit glue, and fish glue) have demonstrated that the thermal decomposition generates small molecular weight compounds that may be linked to a binder type.314,315 Pyrrole ring (C4H5N) was identified, for example, as the main pyrolysis product of Pro and Hyp amino acids, and it is used as a diagnostic marker of animal glue. This heterocycle marker was then used as glue marker during the analysis of the painting layers of two Egyptian objects: the wooden sarcophagus of Usai, Nekhet’s son (XXVI dynasty, 664−525 BC) and the cartonnage containing the mummy of Nekhetubastetiru (XXII and XXIII dynasty, 944−716 BC).316 Other protein markers308 such as diketodipyrrole, indole, and 3methylindole (skatole) were also used. In particular, diketodipyrrole was described as a heat degradation product of Hyp, characteristic of glues. For example, among several thermal degradation products (nitrogen-containing compounds consistent with the presence of proteinaceous materials), pyrrole and diketodipyrrole, the main markers of Pro and Hyp, were identified in a Coronelli’s terrestrial globe (XVIIth century), revealing thus the presence of animal glue as shown in Figure 16.317 Indoles (indole and 3-methylindole) were defined as pyrolysis products of Trp. High contents of indoles indicate the presence of egg or casein, but the differentiation between egg and casein was accomplished using the heat degradation products of lipids components318 and/or carbohydrates.308 It has to be pointed out that various markers were reported in the literature depending on analyzed binders, but also depending on 26


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Table 4. Pyrolysis Products of Ancient and Modern Human Skin Tissues, Calf Skin Leather, Amino Acids, and Dipeptidesa peak no.

characteristic ions (m/z)

19 20 21 22 23 24 25

69, 68, 42, 41 pyrroline 67, 52, 41, 40 pyrrole 92, 91, 65, 51 toluene 95, 80, 67, 44, 53 4-hydroxypyrimidine 81, 80, 53, 40 C1-pyrroles (2) 106, 91 ethylbenzene 104, 78, 51 styrene 95, 94 (80), 80, 67, C2-pyrroles 53 94, 66, 65, 40 phenol 94, 67 pyridinamine 107, 106, 67 p-aminotoluene 113, 84, 56, 42 1 -methyl-2,5pyrrolidinedione 111, 83, 68, 40 ? 95, 68, 41, 40 4-aminopyrimidine 108, 107, 79, 77, 4-methylphenol 66 99, 56, 40 succicimide 117, 116, 90, 89 ethylcyanobenzene 113, 94, 70, 55, 42 3-methyl-2,5pyrrolidinedione 125, 68, 54, 43 ? 131, 91, 78, 66, 51 propylcyanobenzene 120, 91, 66, 51 vinylphenol 122, 79, 52 pyridine derivative 117, 90, 89, 63 indole 134, 119, 91, 65 4-isopropenylphenol 150, 135, 107, 77 vinylguaiacol

26 27 28

131, 130, 103, 77 138, 98, 69, 54, 42 166, 151, 70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

peak no.

characteristic ions (m/z)

Pro Pro Phe

29 30 31

Pro, Hyp Phe Phe DKPs rearrangement Tyr and/or peat

32 33 34

140, 112, 69, 41 152, 100, 68, 41 136, 107, 80, 53, 40 164, 138, 93, 66 178, 163, 94, 42 182, 125, 97, 96, 70 164, 136, 94, 66 168, 125, 97, 70, 44 186, 130, 93, 66 154, 111, 83, 70, 41 172, 171, 143, 65, 52 176, 94, 66, 54 196, 154, 125, 70,41 185, 94, 92, 66, 41 200, 199, 154, 125, 70 192, 191, 70, 68, 41 204, 189, 94, 66 194, 138, 96, 70, 41 195, 154, 125, 70, 41 210, 124, 86, 70, 41

compd name

C1-indole trimethylpyrimidine ?

origin

35 36 37 38 39

Pro-Asp Tyr and/or peat

40 41

asparagine Phe

42 43 44

Pro-Leu Phe Tyr and/or peat

45 46 47

Trp Sphagnum monocotyledon lignin Trp

48

compd name ? ? ?

origin Pro-Arg

? ? ? ? 2,5-diketopiperazine diketodipyrrole pyrrolidinopiperazine derivative 5-hydroxy-4-phenyl pyrimidine ? 2,5-diketopiperazine

Pro-Ala Hyp Pro-Gly

Pro-Arg Pro-Val

? ? 2,5-diketopiperazine

Pro-Hyp

? 2,5-diketopiperazine

Pro, Pro-Pro

2,5-diketopiperazine

Pro-amino acid

2,5-diketopiperazine

Hyp

a

Numbers underlined indicate MW and in bold indicate base peak (see related publication for supplementary details). Reprinted with permission from ref 321. Copyright 1997 John Wiley and Sons.

products of amino acids were the major contributors to the total pyrolysates of all of the skin tissues as shown in Table 4, indicating that proteins in ancient samples were preserved (others products such as 4-isopropenylphenol provide evidence of reactions between amino acids and other components such as sphagnum acid). Applied to archeological samples (approximately 0.15 mg each) ranging from 40 000 to 50 000 years (bone and tooth mineralized tissues from a variety of environmental conditions such as desert, Siberian permafrost, cave, Egyptian tomb),298,322 the pyrolysis GC−MS procedure allowed the identification of several protein markers such as 2,5-diketopiperazines (dipeptide precursors:323 Pro-Ala, Pro-Gly, Pro-Val and/or Pro-Arg, ProHyp, Pro-Pro). The markers of phenylalanine (toluene, styrene, ethylcyanobenzene, propylcyanobenzene) were also detected, as well as Pro markers (pyrroline, pyrrole, alkylpyrroles) and Hyp markers (diketodipyrrole, pyrroline), suggesting the presence of collagen (comparison to samples of collagen and fresh skin). Recently, the 2,5-diketopiperazines pyrolysis products (in particular from the cyclohydroxyproline−proline precursor) were used as a parameter to assess quantities of collagen inside each analyzed archeological bone.324

the type of pyrolyzer used and instrumental settings; thus this analytical approach requires often an adapted library of binder profiles.296 Pyrolysis GC−MS technique is nowadays still used for the study of artwork; however, it is generally combined with other analytical approaches to fully characterize an artwork (pigments and binders). Pyrolysis GC−MS and optical microscopy, SEM with energy dispersive X-ray spectroscopy, micro-Raman, and MALDI-TOF-MS were used to study the original medieval painting technique dating from the XIIth to the XIIIth centuries-painted crucifix and the subsequently applied restoration materials.319 In this case study, pyrolysis GC−MS identified the various synthetic polymers present in the sample, and the organic binders (egg and glue) were mainly identified by MALDI-TOF analysis (see the section related to proteomics). Pyrolysis GC−MS was also used to assess the quality of protein preservation in archeological and paleontological remains.298,320,321 For example, the study of a 25 million year old beetle cuticle fossil showed the presence of 2,5diketopiperazines, that is, the diagnostic pyrolysis products of proteins. Several other pyrolysis products from amino acids such as phenol and methylphenols (markers of Tyr), indole and methylindole (markers of Trp), toluene (marker of Phe), and pyrrole and methylpyrroles (markers of Pro) were also detected. 320 Another example is the study of protein preservation in the tissue of Iron Age bog bodies by comparing its pyrolysis profile with fresh skin profiles (untreated and treated with various vegetable tanning agents).321 Pyrolysis 27


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3.4. Alternative Techniques for the Analysis of Amino Acids or Low Molecular Weight Tags for Protein or Binder Characterizations in Ancient Samples

CZE coupled to MS was also used for the study of gelatin binders, casein binders, and egg yolk binders.334 The device includes a commercial interface between the capillary electrophoresis system and the ESI ionization source of the MS (ionization performed using electric field applied on a metalliccoated needle containing the liquid sample and additional ions resulting in an aerosol; the analytes are ionized via ion/molecule reactions). The working electrolyte buffer and the flow rate were critical parameters (due to the coupling of electrophoretic system to ESI ionization source). Carbonate buffer (15 mmol L−1; pH 9.0) was used as electrolyte, methanol and water (50/ 50, v/v) as a makeup solution, and the best flow rate was 10 μL min−1. Samples were hydrolyzed (40% hydrobromic acid, 60 °C, starting material below 5 mg), and amino acids were analyzed. General profiles of binders were studied as well as characteristic ions of binders using SIM mode analysis (detailed previously). For example, Tyr (m/z 182) was attributed to egg yolk and casein binders, cysteine (m/z 241) to egg yolk, and Hyp (m/z 132) to gelatin. Hyp, Leu, and Ile (amino acids with similar molecular weights at m/z 132 and similar electrophoretic mobilities, but not similar structures nor elementary compositions) were differentiated using an increased ESI voltage (280 V versus 60 V) that produced fragmentations of Leu and Ile. Capillary electrophoresis mass spectrometry was also used for age estimation of silk (B. mori) textiles.335 The amino acids resulting from acid hydrolysis (HCl 6 N; less than 100 μg of silk starting material) were separated using the (+)-18-C-6-TCA background electrolyte. This background electrolyte served also as complexation reagent.329 Thus, D- and L-amino acids were separated and detected via protonated amino acid/18-C-6-TCA complexes. The D/L ratios of Asp were measured during the analysis of several well-dated textiles (∼2500 years ago to present). The effect of the digestion duration of the silk samples on Asp racemization was also studied (long digestion durations cause the conversion of L enantiomer to D enantiomer due to heating). The optimized protocol integrates a digestion duration of 2 h (conversion of L enantiomer to D of 1% versus 4% for 24 h digestion). As a result, the relative increase of the average D/L ratios for silk samples versus the sample age was successively pointed out (Figure 18). Recently, a study focusing on the Asp racemization rate of bones (1−5 mg) from different species (Homo sapiens, Cetacea, Bison antiquus, Hemiauchenia macrocephala, Ursus sp., Equus sp., Cervalces sp., Bootherium sp., Leporidae) using capillary electrophoresis mass spectrometry has provided new insights related to species-specific racemization dating.201 3.4.2. Inductively Coupled Plasma Mass Spectrometry Applied to Artwork Binder Characterization. ICP-MS was proposed to characterize proteinaceous binders of artwork. In this technique, an argon plasma (6000 °C) serves as the ion source (samples are introduced into the argon plasma as aerosol droplets; the plasma dries the aerosol, it dissociates molecules, and ionization is mainly achieved by collisions of energetic argon electrons). The quadrupole (detailed previously) is the commonly used analyzer. ICP-MS is a widely used technique for elemental analysis such as metals (trace analysis at the partper-billion level, i.e., 10−9 g/g); nevertheless, several applications related to protein analysis were proposed.336,337 In particular, ICP-MS was applied to the detection of proteins (hundreds of fmol detected) by probing specific heteroatoms naturally present such as sulfur (in cysteine and methionine residues) or phosphorus (in phosphorylated proteins). Chemical derivatizations were also proposed with the use of chelating agents such as

3.4.1. Capillary Zone Electrophoresis Applied to the Study of Ancient Samples. CZE is an analytical method that separates analytes depending on their electrophoretic mobility. The technique is based on an electro-osmotic flow in a capillary using a background electrolyte under an electric field. Detection can be performed using various techniques (involving or not the derivatization of analytes) such as UV−vis or fluorescence absorbance,325 CCD detector,326 or MS.327−331 CZE was introduced in 2004 for the characterization of proteinaceous binders.332 Collagens, egg white, and milk casein were hydrolyzed (HCl 6 M), and amino acids were subjected to CZE-CCD analysis (no derivatization needed using CCD detector). The selection of the pH of the background electrolyte has represented one of the most important steps as it improves the separation selectivity (it determines the analyte effective mobility). Chloroacetic acid (51.9 mM) with LiOH at pH 2.26 was used. The characterization of standard samples was based on the amino acid composition (using the RPA). The identification of Hyp associated with a large Gly content (RPA > 20%) was shown to be characteristic of gelatin. Differentiation between egg white and casein was based on the Pro content: the RPA value for Pro was described higher than 15% for casein, while it was less than 7% for egg white. Furthermore, the sum of Ser and Val was higher than 20% for egg white and lower for casein. The proposed scheme was tested on a wider variety of proteinaceous binders including different glues (parchment glue, rabbit skin glue), and it was found to be well applicable.332 The study of potential interferences arising from other compounds such as plant gums and drying oils was investigated on several mixtures.333 The electropherogram patterns resulting from the CZE analyses were constant, demonstrating no oil or gum interferences in fresh samples and in 3 year old samples. Thus, CZE showed its capability during the analysis of a polychrome painted sculpture dating from the XVIIIth century (5 mg sample). As a result, the identification of Hyp and the large concentration of Gly confirmed the presence of the glue that was used as adhesive in the filling applied for this statue (Figure 17).

Figure 17. Electropherogram obtained from a sample of a filling from a statue from the 18th century. For comparison, the electropherogram obtained from the hydrolysate of collagen (gelatin) is also shown. See related reference for conditions and abbreviations. Reprinted with permission from ref 333. Copyright 2005 Springer. 28


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Figure 18. Average D/L ratios for silk samples. Silk samples include fresh silk (2010), untreated silk from the Oka Studio (∼1990), a silk flag from 1883 to 8, a silk flag from the Mexican War (1845−6), pink (top) and blue (bottom) silk from a man’s suit coat (1740), silk from the 1540s, yellow (top) and blue (bottom) silk from Egypt (993), and brown (top) and red (bottom) silk from China (475−221 BC). The inset shows the electropherogram of each sample’s aspartic acid D and L peaks (547 m/ z). Reprinted with permission from ref 335. Copyright 2011 American Chemical Society.

DOTA or DTPA. These reagents can be used to label the functional groups of peptides or proteins; they are then chelated with lanthanides (e.g., cerium, samarium).338 Mainly used for quantitative studies in biological applications based on peptides339−341 or proteins,342 the technique was evaluated for the characterization of proteinaceous binders in artwork.343 Model binders based on egg, glue, and casein were studied. In particular, protein labeling was performed with DTPA and chelation with europium. The samples (0.6 mg) were analyzed with HPLC (C18) ICP-MS to obtain the characteristic retention times of proteinaceous binders; more precisely, the peak corresponding to europium was monitored (m/z 151) (Figure 19). Glue, casein, and egg/ovalbumin samples have shown different retention times and profiles (a peak detected at 10 min for glue, around 13 min for casein, and several peaks for egg or ovalbumin between 10 and 24 min). Analyses of two unknown samples (no information provided) (Figure 19) showed similarities to glue models and have illustrated the potential of ICP-MS for binder characterization in artistic samples.

Figure 19. Analysis of pictorial layer samples as compared to (a) animal glue standard, (b) sample 1, and (c) sample 2. For samples 1 and 2, to avoid contamination of the instrument with Eu, from t = 0−4 min of the chromatographic run, the eluent flowed to waste. Reprinted with permission from ref 343. Copyright 2011 John Wiley and Sons.

4.1. Gel Immuno-Based Techniques Applied to the Study of Ancient Samples: Gel Immunodiffusion, Western Blot, and Alternative Techniques

4. PROTEIN IDENTIFICATION BASED ON IMMUNOLOGICAL METHODS: APPLICATION TO ANCIENT SAMPLES Immunological methods are based on the antigen/antibody interaction, offering thus, by the formed complex, a technique with high specificity and sensitivity. Both monoclonal and polyclonal antibodies can be used, depending on antibody availability or unique/multiple epitope(s) to be recognized. The experiments can be performed on various supports such as electrophoresis gel, chromatographic phase, or coated supports, and the detection can be done using different techniques such as radiolabeling, fluorescent tags, or enzymatic reactions. The application of immuno-based techniques to the study of proteins in ancient samples was proposed since the early 1960s.344−347 Nowadays, the most recent applications of these immuno-based techniques are proposing, beyond protein identification, imaging studies of ancient samples.

Gel immunodiffusion relies on the complex formed between an antigen and antibody(ies) placed in an agar gel matrix and resulting in a visible precipitate. The technique can be based on the passive immunodiffusion called the double immunodiffusion, AGID, or Oüchterlony immunodiffusion. For this method, the antigen and antibodies are deposited in two different wells in a gel, and the passive diffusion is performed during 10−48 h. Gel immunodiffusion can also imply the electrophoretic migration of the antigen and antibody(ies) through the agar gel by the action of an electric field; it is called CIEP. Both techniques, AGID348 and CIEP,345,349 were used to study ancient samples; however, CIEP was often preferred. The sensitivity range of CIEP is from 10−4 to 10−8 mg/mL according to the antisera used.350 The technique was initially applied to tempera model paints in the early 1960s showing the capability of the technique.345 The 29


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Beyond the study of artifacts, serum proteins in archeological bones were also studied using gel immunodiffusion technique (Oüchterlony technique).348 Bone samples (100−300 mg) were ground in a mill and proteins extracted using different solutions based on guanidine/HCl and EDTA denaturation (multistep procedure based on several extractions, desalting, and centrifugation to separate nonmineral bound proteins, mineral bound proteins, and collagen). The diffusion of samples and antibodies in the gel was done during one night. An additional step of gel washing as well as protein staining were performed. Commassie blue R, Commassie blue G 250, and silver nitrate stains were used and evaluated. Among the 150 archeological bones studied from various sites and epochs (up to 5500 BC), one-quarter gave positive immunological reactions to albumin, A2HS-glycoprotein, and transferrin antibodies as shown in Figure 20 for transferrin. This study showed that serum proteins

antiserum was produced by injection of whole egg yolk in rabbit, and the generated antibodies were used for testing. CIEP allowed the detection of four proteins from egg yolk (four precipitates observed) in the dried paint film. However, the experiments based on the tempera paint dating back 25 years have not shown success. Subsequent applications of CIEP were related to the detection of blood protein residues in various types of archeological materials. For example, antisera (polyclonal antibodies) were successfully used to screen the presence of blood on artifacts of the late Pleistocene−early Holocene age from a boreal forest.349 In this study, the residues from artifacts were removed with 5% ammonia and deposited with the antiserum in agar gel for CIEP experiments. Complementary to the appearance of the visible immunoprecipitate formed by the Ag/Ab complex in the gel, a subsequent gel washing to remove the non complexed proteins (antigens and antibodies) and an additional staining with Coomassie blue were performed on the Ag/Ab complex trapped into the matrix. Beyond the identification of blood on archeological objects, the polyclonal antisera may identify animal species or family.349,351,352 For example, antisera from bison, elk, deer, antelope, mouse, rabbit, rat, quail, trout, and human were used to detect the preservation of blood residues on objects dated over a 5600 year period.351 The antisera of bison, antelope, and trout were described as species-specific; however, the other antisera tested have crossreacted with blood residue of several species351 (e.g., the deer antiserum reacts positively to other animals from the Cervid family such as moose or elk349). Clear evidence for Equidae and Bovidae hunting was also obtained from residues on Paleoindian lithic points (dating from 11 000 to 11 300 BP),353 and Cervidae blood was identified during the study of a large number and a large variety of stone tools (130 objects analyzed) from an early Paleoindian site (ca. 11 200 BP, pertaining to the Gainey phase), indicating that cervids were important resources of Gainey phase population.354 Another example showed that blood was used as binding medium in a chumash indian pigment cake dating from the XIXth century;355 in particular, human, pronghorn, and deer species were positively tested using their respective antisera. Where pronghorn was positively identified because of its speciesspecific antiserum, antibodies from deer have shown cross reactivities with different famililes such as Cervidae or Antilocapridae. The presence of blood from fishes was also identified on the early Neolithic artifacts from an archeological site located outside the town of Malmö in southern Sweden.356 In particular, antisera from American eel, alewife, Atlantic croaker, bay anchovy, catfish, gizzard shad, striped bass, sturgeon, trout, and weakfish were studied (among other tests with antisera from bear, bison, bovine, cat, chicken, deer, dog, elephant, goat, guinea pig, horse, human, mouse, rabbit, rat, sheep, turkey). For example, a positive test for both Atlantic croaker and alewife antisera had suggested the use of the archeological tools for processing spiny-rayed (percoid) fishes and members of the Clupeidae (herring family).356 Recently, the combination of CIEP and typological data gave new information related to the Mahaffy cache (in Boulder, Colorado): it is possibly a Clovis sites (the Clovis culture is a prehistoric PaleoIndian culture dating from 11 500 BC).357 Among the 83 studied artifacts, four were positively tested against sheep, bear, horse, and camel taxons independently. The results related to horses and camelids have thus indicated that the cache is dated from the late Pleistocene. The study has contributed to an increased knowledge related to the animals hunted by Clovis groups in North America.

Figure 20. Detection of transferrin in a medieval bone by immunodiffusion. Samples from Tall Mumbaqa (TM), Schleswig (SL), and Ofnet cave (OF). Left: Test for A2HS, all negative. Right: Test for transferrin (TF), positive for Schleswig (grave 233). Wells: 1, fresh human serum; 2−6, serum protein extract from the bone, concentrated and diluted 1:2, 1:3, 1:4, and 15, respectively; X, antitransferrin. Reprinted with permission from ref 348. Copyright 1998 John Wiley and Sons.

may be well preserved in archeological bones. Besides passive immunodiffusion, the Western blot technique348 was also evaluated successfully during this study.348 The Western blot technique is based, in a first step, on protein separation using SDS-PAGE gel electrophoresis (which separates proteins according to their molecular weights through a polyacrylamide gel and using an electric field). In a second step, 30


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irreversibly some proteins such as collagen and albumin,370,371 or a membrane where samples are deposited (e.g., polyvinylidene difluoride membrane)360 (see details below related to DIBA coupled to autoradiography detection, and Figure 22). Radioimmunoassay was also applied to study fossils. RIA was used to detect the collagen in human bones from different ages dating back to 1500 years372 using a polyclonal antiserum against human serum proteins and starting from demineralized saline extract of bones (5 g of starting material; saline extraction with NaCl and tris (hydroxymethyl) aminomethane at the respective concentrations of 0.147, 0.01 M, pH 7.4; and demineralization with acetic acid 0.09 M or HCl 0.1 M). Further analytical confirmations were based on cation exchange chromatography and the detection of the ninhydrin-derivatized amino acid contents resulting from the total hydrolysis (HCl 6 M) of the purified saline insoluble materials. The identified amino acids were similar to collagen ones, confirming thus the RIA results. In this study,372 additional experiments to characterize the serum proteins that were potentially present in samples remained unsuccessful (e.g., passive immunodiffusion against a polyvalent antiserum to human serum proteins). Radioimmunoassays have furthermore allowed the detection of collagen in avian and mammalian fossils from several Pleistocene sites in Australia (bones demineralization with EDTA 0.2 M and acetic acid 0.5 M).373 The antiserum used was a rabbit antibody against chicken collagen (due to its high reactivity against denatured collagen of any antiserum tested); that is, strong reaction obtained with chicken collagen, 70−80% of that reactivity with emu, cassowary, or rhea collagens, and less than 50% of that reactivity with various mammalian collagens. The presence of osteocalcin was also probed by RIA in an extinct class of New Zealand ratite birds, the moas.374 Formic acid extracts of bones were used in this case as well as antisera for the sheep osteocalcin (antigenic determinant directed against the region 15−49 of the osteocalcin molecule, similar to several species). Figure 21B shows serial dilutions of moa bones extracts

a protein transfer from the electrophoresis gel to a membrane (commonly nitrocellulose) by application of an electric field (electroblot) is performed. Protein detection can be done using different techniques such as a second blot with a secondary membrane, which carries the antibody (commonly cellulose acetate membrane).348 Another detection method is the incubation of the nitrocellulose support in a staining solution that reacts with enzymes and converts the stain in a visible colored precipitate (see details in the section related to enzyme immunoassays).30,358−360 GIA was also used for staining antigens trapped in the nitrocellulose membrane.361 The first step of the GIA technique consists of the use of a primary antibody that binds to the antigen that is confined in the nitrocellulose membrane. In a second step, the gold conjugated second antibody is applied to the membrane, and the second antibody reacts with the serum protein of the first antiserum. The gold bound to the antibody is weakly colored; however, the staining can be improved using silver ions in reducing conditions. However, the technique had highlighted negative results from experiments aiming to detect blood traces on archeological lithic tools.361 Despite the capability of immunogel-based techniques, several cautions in the application of the experimental procedures362 and the interpretation of the results363 were pointed out (e.g., buffers used during sample preparation, careful handling of artifacts, routine tests of soils, tests of antibodies on models, study of false positive),362 and an unsuitable procedure for sample preparation and data interpretation may result in negative results (whereas proteins maybe present) or its opposite (false positive). It has also to be taken into consideration that surviving proteins may be modified, and it may induce decreases in the immunoactivities.364,365 Some comments on the lack of reproducibility of analyses, reliability of identifications, and inaccuracies concerning interpretation of data are described in the literature366−368 as well as their opposite; that is, techniques provide highly valid identification results from ancient residues.350,362 4.2. Radioimmunoassay for Protein Detection in Fossils and Archeological Materials

The RIA technique (or pRIA) is based on the use of radioactively labeled antigens. Commonly, 125I is used because iodine atoms can be introduced into Tyr of protein backbones. The principle is based on competitive binding: a radioactive antigen competes with a nonradioactive antigen for a fixed number of antibodies. Measurements are performed in a scintillation counter by monitoring complexes formed by antigen, labeled antigen, and antibody. A ratio is calculated between the complex formed by antigen, labeled antigen, and antibody (B) and the complex formed by labeled antigen and antibody (B0) (through the radioactivity of the labeled antigen). When the cold antigen (standard or unlabeled sample) is introduced in a system containing a fixed amount of labeled antigen bound to a known amount of antibody, the amount of the labeled antigen bound to the antibody decreased by the increasing amount of the unlabeled antigen. The data obtained for antigen standards serve to construct a dose−response curve, and the quantification of unknown antigens from the samples is then possible. Radioimmunoassay allows the detection of lower amounts than nanograms of antigen.369 The technique is commonly performed in the liquid phase; however, solid phase supports were also developed for Cultural Heritage applications, such as a polyvinyl microtiter plate, which binds

Figure 21. Osteocalcin radioimmunoassay, showing (A) the specificity of the assay; ● = sheep OC, ■ = sheep OC 15−49, ▼ = pig OC, ▲ = wallaby OC, ○ = sheep OC 40−49; (B) displacement characteristics of desalted, lyophilized formic acid extracts of moa bones shown in Table 1; ● = sheep OC, ○, ■, □, ▼, ▲, and △ = samples 1−6, respectively. The ordinate is in units of ng/mL for the sheep OC standard curve and in units of mg equivalent of bone/mL for the bone samples. Reprinted with permission from ref 374. Copyright 1985 Elsevier.

in the sheep osteocalcin radioimmunoassay. Otherwise, osteocalcin was also detected in fossil bovid bones from 12 000 to 13 million-years-old (EDTA treated bones) by the antigen of bovid origin.375 Radioimmunoassay was used in fossil samples to demonstrate the species-specificity of detected proteins,370,376 depending on 31


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Figure 22. Dotblot immunobinding assay of recent and fossil brachiopods. Samples are blotted onto the filter in a row of four halving dilutions, and the membranes are then reacted with large volumes (>30 mL) of test antiserum at low concentration, in solutions containing an excess of serum proteins including unrelated immunoglobulins. The immunological signals are detected by autoradiogram. A range of samples and sera are shown to illustrate the criterion of systematic specificity. Normal Rabbit Serum (NRS; i.e., a serum taken prior to immunization with brachiopod shell macromolecules) is a standard immunological control. α-Notosaria, a serum raised against a rhynchonellid (distantly related to all of the samples tested), is an outgroup control. α-Liothyrella is a serum raised against a genus, which belongs to the same family as Pliothyrina sp. (F. terebratulidae). α-Waltonia and α-Dallina (F. Terebratellidae) belong to the same family as the genera Neothyris, Terebratella, and Waiparia, and Pachymagas mercenaria (a 70 Kyr fossil bivalve mollusc) is used as an outgroup antigen. A reaction that is significantly greater than NRS is commonly regarded as positive; using this sole criterion, all brachiopod samples give positive results. However, by comparing the systematic specificity of the immunological reactions with those of recent genera (Figure 1), only the Plio-Pleistocene Neothyris and Terebratella are considered to yield significant immunological reactivity (see Table 2 for a full summary). Ages (in Myr) are estimated from stratigraphic position of the sampling localities (Table 1). Mg shell equivalent of the volume of EDTA extract of each antigen is given to the right of the figure. In recent shells, 0.0014 mg is equivalent to approximately 1 ng of organic matter. Reprinted with permission from ref 360. Copyright 1991 Elsevier.

acetic acid extraction).370 In particular, the bone material from an Egyptian mummy radius (dating back 3000 years) and a Homo erectus mandible (dating back 500 000 years) was used to elicit antibodies to human collagen. The radioimmunoassay technique showed also its abilities to discriminate human bone fragments from animal bone fragments (fragments too small to allow morphological species identification,383 or in a radioimmunoassay-blind test context).384 For example, antisera to human, bison, bear, rat, elephant, and elk albumins were used.383 Radioimmunoassay was successfully applied to the study of shells from Plio-Pleistocene (both Pliocene and Pleistocene epochs) using antibodies against the biopolymers from a range of recent brachiopod shells to reconstruct phylogenetic relationships.360 Starting from EDTA-based demineralized samples, the dot-immunobinding assay (Figure 22) was used according to the following procedure: (i) samples were deposited on a polyvinylidene difluoride membrane as a small dot; (ii) the membrane was then incubated in a first antiserum, this antiserum was prepared against the skeletal biopolymers from modern brachiopods, and the immunoglobulin G fraction was partially purified by ammonium sulfate precipitation; (iii) the membrane was then incubated in a second 125I-labeled serum that recognizes the first antibody; and (iv) an autoradiography was finally performed (Figure 22) (the alternative technique described consists of the use of alkaline phosphatase labeled goat-anti rabbit IgG as second antiserum and visualization of an insoluble colored precipitate (bromochloroindolyl phosphate/ nitrotetrazolium substrate)). As a result, the immunological reactions for ancient and modern samples were positive (except for the samples older that 4 million years) showing the successful

protein survival (highly dependent on several conditions such as burial or climatic conditions).377 For example, the technique was used (in a complementary way to immunodiffusion assay) to detect serum albumin in muscle from a 40 000 year old mammoth (Mammuthus primigenius).378 The frozen muscle was ground, suspended, and injected into rabbits to produce antibodies. The mammoth antiserum reacted strongly with elephant albumin, weakly with sea cow albumin, and more weakly (or not) with other mammalian albumins tested. This study had pointed out the similar antigenic sites between the native mammoth and the elephant albumins. Elephant-like proteins were also found in bones of mastodon: antibodies elicited from mastodon bones or mammoth muscles have reacted strongly with the collagen and serum proteins of extant elephants.379 Immunological comparisons were thus provided: mammoth was found to be closer to Asian and African elephants than to the mastodon, whereas mastodon was closer to these elephant species than to mammals outside the Proboscidea order. Albumin was furthermore detected in two recently extinct species, the Siberian mammoth and the Tasmanian wolf.380 In particular, based on the radioimmunoassay data and thus the immunological similarities between species (e.g., Tasmanian wolf and related species), the albumin phylogeny was proposed. Another example showed that albumin from skull fragments dating back over 1.6 million years was found immunologically closer to human albumin than equid or bovid albumins.381,382 This result supports the theory that Hominidae were living in Andalusia at this period. Human fossils dated up to two million years, such as fossils from Australopithecus robustus, were also studied using their collagen and albumin contents (EDTA and 32


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period). Antigens were extracted from samples using a tris (2amino-2-hydroxymethylpropane-1,3-diol) buffer saline solution, and they were coated on nitrocellulose membrane for antibodybased detection.359 Antisera against human globin (apohemoglobin), the α-chains and the β-chains, were used to detect hemoglobin in bones of ancient Roman, Iron, and Eneolithic ages.387 The antigen from bones was extracted using sample grinding in nitrogen, protein extraction with urea, protein purification using gel filtration chromatography, lyophilization, and tris buffer saline solubilization. Human antibodies antiimmunoglobin G were used to detect human blood on artifacts dating from 100 000 years ago.30 Enzyme immunoassay was also successfully used, in parallel with radioimmunoassay, to study Plio-Pleistocene shell proteins (from EDTA-demineralized shells) deposited on polyvinylidene difluoride using antisera raised against intracrystalline macromolecules from recent brachiopod shells (see details in the section corresponding to radioimmunoassay).360 Quantitative dot-blotting assays were also proposed to study immunoglobulins from 1.6 million year old fossil bones (PBS, EDTA, and acetic acid extracts) using orthophenylenediamine dihydrochloride peroxidase detection; that is, nitrocellulose membrane spots were cut and the absorbance was read at 490 nm with a microplate autoreader.389 Applied to the study of proteins extracted from the skeleton of a mastodon from northern Venezuela (EDTA protein extraction), SDS-PAGE gel electrophoresis (see the related section for technical details on Western blot) complemented by a Western blot has shown a 68 000 Da band by immunodetection, corresponding to albumin in its intact form.358 Immunoblot following an isoelectric focusing experiment (separation of proteins according to their isoelectric point on a polyacrylamide gel using an electric field) was also successfully used to study α2HS-glycoprotein and α1-antitrypsin from ancient Peruvian human bone samples (115−1450 AD).390 Among the different enzyme immunoassay techniques and as introduced previously, ELISA represents the most commonly used technique to study proteins from ancient samples,375,391 but also to establish relationships between species (e.g., relationships within the terebratulid brachiopods)392 or the diagenesis/degradation processes.393−395 For example, polyclonal antiosteocalcin antibodies from purified bovine and rat osteocalcins (horseradish peroxidase-linked used as second antibody) were used to detect osteocalcin, respectively, from fossil bovid bones dating from 12 000 years to 13 million-yearsold, and in rodent teeth dating from 30 million-years-old.375 Another example is the detection of hemoglobin in skeletal remains from Roman and Medieval cemeteries.396 Hemoglobin was obtained from human blood by salt fractionation (ammonium sulfate), and a subsequent purification step, using a sepharose column, was used to separate albumin from hemoglobin (and antialbumin from antihemoglobin) and thus reduce the problems of crossreactivity associated with polyclonal antisera. Monoclonal antibodies were also evaluated against human albumin.397 Despite the poor skeletal preservation, the presence of human albumin was identified in bones from both medieval subjects studied and in 9 of the 12 Saxon individuals as shown in Figure 23. Further investigations using albumin monoclonal antibodies showed positive results in bone extracts from Bronze Age human remains (2200−1700 BC) as well as in skeletons from English Civil War (AD 1644), Roman (AD 100− 200), and Iron Age (ca. 400 BC) burials.398 No evidence of cross-reactivity with animal material was shown, but the detection seemed to depend, beyond the sample amount, on

detection of protein macromolecules from shells dating back two million years using immunological analysis. Osteocalcin was also successfully detected in dinosaur bones and fossils from other vertebrates (e.g., Pleistocene, Miocene).385 The presence of osteocalcin was confirmed by detection of the γ-carboxyglutamic acid bound to the osteocalcin (following its alkaline hydrolysis) using reverse phase HPLC. The study also showed immunological cross-reactivities with modern samples and young fossils using the antibodies raised against osteocalcin from modern vertebrates. Overall, the study had provided new information toward the phylogeny of dinosaurs. Radioimmunoassay was also evaluated for the study of bloodstains383 or other protein residues369 on lithic artifacts. However, applied to faunal remains and stone tools from two sites from arctic and subarctic contexts,386 the technique showed misidentification and cross-reactions due to diagenetic alteration of proteins.386 4.3. Enzyme Immunoassay and Enzyme Linked Immunosorbent Assay Technique for Protein Identification in Art, Archeology, and Paleontology

The EIA technique is based on the recognition of the antigen attached to a surface, using an enzyme-labeled antibody to form a complex antigen/antibody (Ag/Ab) involving a reaction catalyzed by an enzyme and resulting in a colored/fluorescent conversion of a chemical that is added in the reacting system. ELISA is an EIA technique, and it is one of the most common tests in immunobiochemistry. Different ELISA techniques or designs are proposed. For example, direct ELISA consists of direct reaction between the enzyme-labeled primary antibody and the antigen bound to a microtiter plate, whereas the sandwich ELISA (indirect ELISA, the most commonly used technique) detects the antigen bound on a plate coated with a capture antibody, using another antibody (i.e., the antigen is trapped between the two antibodies), and the enzyme-linked secondary antibody is added to detect the first antibody, and finally, a substrate is added and converted by an enzyme to its detectable form. 4.3.1. Enzyme Immunoassay and Enzyme Linked Immunosorbent Assay and Derived Techniques Applied to Archeological and Fossil Materials. EIA was introduced at the mid/end 1980s to detect proteins in ancient samples using an enzyme-labeled antibody.30,358,387,388 As described previously, among the different techniques characterizing enzyme immunoassay is found the detection of the antigens deposited (DIBA) or electroeluted (resulting from Western blot experiment) on a membrane such as polyvinylidene difluoride360 or nitrocellulose.358,387 As mentioned previously, protein detection consists of the incubation of the membrane in a stain that reacts with enzymes (labeled to the antibody) and converts the stain into a visible colored precipitate; for example, the 4-chloro-1naphthol stain is converted by the action of the horseradish peroxidase enzyme (conjugated with the antibody) to a purplecolored precipitate.358 Another example is the use of the alkaline phosphatase enzyme with bromochloroindolyl phosphate and nitro blue tetrazolium that produces a bluish-purple product.360 As mentioned previously, the technique can be based on the use of unique enzyme-labeled antibody or two antibodies, the first directed against the antigen trapped in the membrane and the second directed against the first antibody.359 Picograms of antigens can be detected by the technique.30,359 For example, polyclonal antibodies were successfully used to detect blood from lithic artifacts from a Paleoindian site (late Pleistocene 33


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survival.405 Water was shown to be the major factor for diagenetic changes in proteins, more than age, soil content, or pH. Bone integrity was also shown to be an important parameter for the survival of proteins.405 Besides, the role played by temperature on the survival of blood proteins was pointed out using analysis of cremated materials (e.g., skeletal remains of cremated individuals, cremated ceramics containing human serum).406,407 The degradation rate of osteocalcin from ancient bones408 was further investigated. Monoclonal antibodies directed against three different regions of osteocalcin (Nterminal His4-Hyp9; C-terminal Phe45-Val49; midregion Pro15Glu31) were used during the study of the temperature-stressed modern bone powder.409 In particular, the importance of mineral association to protein survival was shown; for example, the immunological data have indicated a variable survival of the γ-carboxyglutamic acid-rich midregion epitope of osteocalcin in Neolithic bones. ELISA (polyclonal antibody) was also used to detect the relative amount of the remaining osteocalcin in bones in a complementary way to the measurement of several diagenetic parameters (e.g., mineral changes, porosity, material histological preservation);410 in particular, the microbial taphonomy and the mineral alteration of bone were shown to have a damaging effect on the preservation of the osteocalcin. However, it can be pointed out that osteocalcin was successfully detected in dinosaur bones using a set of immuno-based techniques (such as radioimmunoassay,385 see the related section for details), including ELISA. For example, osteocalcin was detected in an 80 million year old Campanian hadrosaur, Brachylophosaurus canadensis,411 using ELISA but also using complementary immuno-based techniques (e.g., immunohistochemistry; see the related section for details). The collagen I protein was also successfully detected using ELISA (and additional techniques) from the Tyrannosaurus rex fibrous cortical and medullary tissues.148 Derived from the ELISA methodology, DACIA was proposed for the analysis of mineral-bound proteins.412 DACIA is based on the use of hydrofluoric acid allowing sample demineralization and the simultaneous capture of proteins onto the solid phase for immunological assay. Using antibovine casein monoclonal antibodies, the technique was successfully applied to detect casein in ethnographic pots from the Punjab region of Pakistan and Gujarat in India (used in 1997 for processing milk), whereas previous attempts using conventional extraction methods failed.412 Successful detection of the αs1-casein of bovine milk was also shown in several cooking vessels dated from the middle of the first millennium BC,413,414 demonstrating that human farming activities were already in place in the studied environment (Cladh Hallan, on South Uist in Scotland). 4.3.2. Enzyme Linked Immunosorbent Assay Applied to Paint Media Analysis. The potentiality of ELISA was first evaluated on collagen-based artwork in the 2000s.415−420 The complex matrix in which proteins are trapped (inducing potential interactions between proteins and other materials such as pigments418,420), the degradation state of the protein material, as well as sample amounts available for analysis make the application of ELISA technique to ancient artwork challenging.418,420 The first step of the ELISA procedure consists of a protein extraction from the sample (starting from several hundreds micrograms of samples). Protein extraction can be performed using different solutions. The sample may be ground and dissolved in buffers (e.g., ammonium bicarbonate),421 and the extraction buffer can contain acids (e.g., trifluoroacetic acid,422 HCL231) or denaturating agents (e.g., trifluoroetha-

Figure 23. Results obtained with fresh human (FBH), Saxon ancient human (AB152 and AB28), fresh ox (FBO), and pig (FPB) bone extracts when tested against doubling dilutions of monoclonal antihuman albumin. The diminished color development seen with human (++/+) as opposed to animal (−) extracts denotes the presence of human albumin. Reprinted with permission from ref 397. Copyright 1990 Nature Publishing Group.

the chronological age. Monoclonal antialbumin was also successfully used to study a 1.6 million year old fossil from Venta Micena in Orce (complementarily to radioimmunoassay).381,382 Immunological detection of another blood protein, immunoglobulin G, was investigated using monoclonal antibodies, but this protein was detected only once among 31 samples (from the English Civil War, medieval, Early Saxon, Roman, Iron Age, and Bronze Age periods), analyzed in parallel with albumin (monoclonal antibody) that was detected in 21 of these samples. This study confirmed that albumin is a better target molecule for protein detection in skeletal remains.399 Monoclonal antibodies against human alkaline phosphatase were successfully used during the study of ancient Egyptian bones from the ptolemeic period (305−30 BC).400 ELISA was also used to study the immunoreactivity of alkaline phosphatase (polyclonal antibodies were used) in ancient bone samples from 429−664 AD and XIIIth−XVIth centuries;401 this information was coupled with further investigations on the binding activities of alkaline phosphatases. Among other target proteins studied is described dermatopontin, a mollusk shell matrix protein, detected in 1500 year old fossils of the extinct land snail Mandarina luhuana by significant immunological reactivity with antiserum raised against a type-1 dermatopontin fragment (partial amino acid sequence near the C-terminus) of the living land snail Euhadra brandtii.402 The materials extracted from a well-preserved 100 000−300 000 year old mammoth skull were also used to produce antisera whose specificity for binding to elephant or mammoth was successfully tested by ELISA and Western blot issued from SDS-PAGE experiments;403 however, the chemical nature of the antigens was not elucidated. Another study used both monoclonal and polyclonal antibodies to detect plasma proteins such as α2-HS-glycoprotein, albumin, and α1antitrypsin in human bone samples from a Peruvian site dating from 115 to 1450 AD.404 The study showed nonspecific reactions with both polyclonal and monoclonal antibodies. The nonspecific reactions of ancient samples were potentially attributed to diagenesis; however, several nonspecific reactions were also detected with modern animal bones adding a cautionary note to immunological approaches toward the study of ancient proteins. For a better understanding of protein survival in ancient fossils, several factors that may influence protein preservation were studied. ELISA analyses based on antibovine albumin monoclonal antibodies were carried out on intact and broken bones to determine the environmental factor roles in protein 34


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Figure 24. ELISA result for the identification of the binding medium on Saint John (see Figure 1 of the related article). On the left is the result for a scraping of the gesso layer and the gilding adhesive immediately below the metal leaf. On the right is the result for a scraping of only the gesso layer beneath those two layers. To confirm the presence of the antigen, four dilutions were prepared from each sample, one for each antibody: ovalbumin (egg), casein (milk), collagen (animal glue), and polysaccharides (gums). Both ovalbumin and collagen are clearly present in the first sample, while only collagen is present in the second. Thus, the binder for the gesso is animal glue, and the adhesive for the gilding is egg. Reprinted with permission from ref 418. Copyright 2010 Springer.

nol).422 Extraction can be also done by the use of denaturing agents (e.g., urea) with detergents (e.g., sodium dodecyl sulfate) and chelating agents (e.g., ethylenediaminetetraacetic acid) in a mixture.416 The extraction procedure may also use sonication of the sample in a buffer (e.g., phosphate buffered saline).423,424 In all cases, protein extraction is crucial because it gives access to protein epitopes for further antibody-based detection. ELISA applied to artwork is commonly based on the indirect ELISA procedure (see details previously); the primary antibodies used for protein detection in artwork are monoclonal420 or polyclonal422 antibodies, usually directed against collagen, casein, or ovalbumin that represent the main binders or adhesives used in artwork. The technique based on enzymelabeled secondary antibodies includes the use of the alkaline phosphatase activity416,420 that allows the colorimetric detection by the enzymatic reaction of p-nitrophenyl phosphate to pnitrophenol. The other detection system coupled to the secondary antibody is based on horse radish peroxidase allowing the colorimetric detection by the enzymatic reaction of 3,3′,5,5′tetramethylbenzidine to 3,3′,5,5′-tetramethylbenzidine diimine or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) to its radical cation.422 The use of a blocking agent before the addition of the primary antibody is generally used to reduce the background due to antibody unspecific binding, or to its crossreactivity with different proteins with similar epitopes; salmon serum in PBS called sea block is for example commonly used, but also newborn calf serum or nonfat dry milk.416,420,422 Applied to the study of a sample taken from a sculptural element of the XVIIth century French cabinet from the J. Paul Getty Museum collection, ELISA with antiovalbumin IgG (hen egg white) primary antibody generated in a rabbit and an antirabbit IgG alkaline phosphatase-conjugated secondary antibody (generated in a goat) had positively reacted, confirming thus the presence of egg white.416 In this study, where ELISA (in

a way complementary to immunofluorescence microscopy, see details in the next paragraph) succeeds in confirming the identification, the presence of egg white was suspected using FTIR and GC−MS but not fully confirmed due to difficulties in data interpretation. Polyclonal antibodies against egg white, milk, and animal glue were also successfully used, in a way complementary to monoclonal antibodies against plant gums (and other techniques such as FTIR and GC−MS) to study grounds and media from Egyptian cartonnage fragments in the Petrie Museum (Ist century AD).425 Positive detections of egg white (but the presence of egg yolk was not excluded) and animal glue (and plant gum) were provided independently in the different samples, whereas no evidence of the use of milk (via casein detection) in these samples was shown. The ELISA technique was also successfully proposed for the identification of several proteins, that is, ovalbumin, collagen, casein, and polysaccharides in different museum objects of the Metropolitan Museum of Art collection (New York).418,422 For example, applied to the study of the Saint John polychrome statue dating from the late XIIIth century, collagen was detected in the gesso layer and ovalbumin and collagen in the gesso layer and gilding (Figure 24), confirming thus that animal glue is the binder for the gesso and egg is the adhesive for the gilding.418 Further studies related to aging effects (47 days artificial aging with xenon-arc weatherometer, with filters to stop far UV-light, 0.5 w/m2 irradiance, 60−80% humidity, 40 °C) and the presence of pigments (e.g., charcoal, red ochre, Lapis Lazuli, maya blue, lead white, chalk, malachite, green earth, vermilion) on immunological detection of proteins (ovalbumin, casein, and collagen) were investigated.420 The ELISA technique allowed successful detection of casein and egg in all aged paints; however, experiments with glue have resulted in signal loss. On the basis of experiments involving monoclonal antibodies against bovine βcasein and chicken ovalbumin, the effect of pigments (azurite, 35


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smalt, Lapis lazuli, minium, lead white, hematite, cinnabar, giallorino, verdaccio, manganese black, umber, malachite, zinc white) on antigen detection was not observed from fresh paints (15 pg of milk and about 7 ng of albumen detected for the dried binders).423 However, analyses of the artificially aged paints have suggested some degradation processes or masking effects involving the recognized epitope; for example, sensitivity modifications of experiments based on β-casein paints (including various pigments such as smalt, minium, lead white, hematite, malachite, cinnabar, giallorini, Mn black) and lead white-ovalbumin paints. However, positive detection of β-casein and chicken ovalbumin was shown independently in different samples of the transept vaults of the upper church of the Basilica of St. Francis in Assisi (Italy). The chicken-egg yolk monoclonal antibody was also successfully tested in a microsample from “Polittico di S’Angelo” (1499) by Niccolò Liberatore; the sample was furthermore tested for ovalbumin and casein, but these proteins were not detected, indicating that the binder was egg yolk and not whole egg.424 ELISA experiments are generally used to screen the presence of a target protein in a sample, but it is also used as complementary technique to complete/confirm results from other analytical techniques such as GC−MS (analysis of amino acids mixtures resulting from the hydrolysis of the sample) or liquid chromatography-electrospray ionization-tandem MS (analysis of the peptides resulting from enzymatic hydrolysis of the sample; see details in the proteomics section); these techniques as well as fluorescence were, for example, successfully applied in a recent study of RomanoEgyptian panels in the collections of the J. Paul Getty Museum dating to 180−200 AD.421 In particular, animal glue was detected in the ground layers of the studied panels using ELISA, and the results were confirmed using MS-based techniques. Mixtures of egg and glue were also detected using ELISA in several of the studied samples and confirmed using the complementary analytical techniques. Dot-ELISA (dot immunoblotting assay) was proposed as a semiquantitative method for the study of egg-based paintings.231 In the proposed procedure, samples are deposited on a poly(vinylidene fluoride) membrane231 or nitrocellulose membrane,426 and detection was provided using the antichicken egg primary polyclonal antibody (developed in rabbit), and antirabbit IgG alkaline phosphatase was used as secondary antibody. Colorimetric detection was performed using 5-bromo4-chloro-3-indolyl phosphate with nitro blue tetrazolium. Alkaline phosphatase hydrolyzes 5-bromo-4-chloro-3-indolyl phosphate to 5-bromo-4-chloro-3-indoxyl that is oxidized by the atmospheric oxygen to form 5,5′-dibromo-4,4′-dichloroindigo (blue dye), and by nitroblue tetrazolium, which forms diformazan precipitate after reduction (dark blue). To attempt to obtain semiquantitative data, samples were analyzed using a spectrophotometer with an integrating sphere (enabled collection of light scattered by the whole sample surface) in the visible wavelength range (380−780 nm). The detection limit for egg white samples had reached 0.5 μg mL−1 (2.5 ng antigen per spot) and for whole egg 1 μg mL−1.231 Applied to the study of three samples from the Giotto “St. Francis receiving the Stigmata” wall paintings in the Basilica of Santa Croce in Florence, the identification of ovalbumin was achieved for two painted samples (less than 1 mg of starting material), while no reaction was observed for the sample taken in the frame around the painting (Figure 25).231 Applied to mural paintings of Saint Francesco Church in Lodi dating from the XIIIth−XVIth

Figure 25. Results from dot-ELISA of Giotto’s “S. Francis receiving the Stigmata” samples with blank, negative, and positive controls. The meaning of A, B, and C is explained in the text (see related reference). Reprinted with permission from ref 231. Copyright 2012 Springer.

centuries (100 μg of starting material), a dot-blot immunoassay showed also the successful detection of ovalbumin.426 4.4. Immunofluorescence Applied to Ancient Samples

In the 1980s, immunofluorescence was proposed to study proteins in various paleological,427,428 archeological, and artwork samples.429 IFM is based on the use of an antibody chemically linked to a fluorophore that can be detected using fluorescence microscopy. Direct or indirect immunofluorescence methodologies can be used. The direct methodology uses a single primary antibody bound to the fluorophore, whereas the indirect technique needs a primary antibody forming a complex with the target antigen, and detection of interaction is ensured by a secondary antibody conjugated to a fluorophore. Among the most commonly used fluorophores are rhodamine B (excitation wavelength range 525−540 nm (green); emission wavelength range 615−630 nm (red)) and FITC (excitation wavelength range 490−495 nm (blue); emission wavelength range 520−530 nm (green)). One of the main advantages of the technique is the possibility to obtain the spatial distribution of the antigen in the studied sample. Detection of two antigens in the same sample using rhodamine and FITC labeled antibodies (directed against two different primary antibodies) is also possible.416,420 Considering artwork, the technique was introduced to detect egg white in paintings.429 FITC fluorescent dye was used as a tag of the secondary antibody (excitation wavelength range used in the study about 420−480 nm). Except for the artwork samples that showed the autofluorescence disturbing the immunofluorescence results (e.g., artwork including lead/zinc white pigments; artwork including natural resins, oils, gelatin glues), the method was successfully applied to cross-sections from paintings and polychrome sculptures dating from the XIIth to the XXth centuries. For example, the experiments carried out on browns and blues from the Mystic Lamb (van Eyck) revealed that some glaze-like layers with ultramarine contain egg white.429 Further protocol optimizations on model paints were proposed for immunofluorescence application to ancient artwork.430 For example, the use of trypsin (endoprotease that hydrolyzes proteins at their carboxyl side of the Lys or Arg amino acids, except when they are followed by Pro) was proposed as pretreatment to make the epitope more accessible to antibodies. The use of a blocking agent (nonreactive proteins) to reduce the nonspecific protein binding was also proposed, that is, 10% horse 36


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Figure 26. (a) Normal light and (b) fluorescence confocal microscope images (100×) of a cross-section of an artificially aged painting layer of lead tin yellow in egg on a gypsum/glue preparation. Inset (c) shows the laser scanning time-resolved immunofluorescence image corresponding to the area evidenced by red contours in (a) and (b). QD605 fluorophore was used to tag egg ovalbumin. The fluorescence confocal microscope image was acquired by exciting at 458 nm and collecting from 595 to 620 nm. The time-resolved immunofluorescence image was obtained at 400 nm of excitation wavelength at 9 ns of time delay (laser pulse width of 60 ps). The image is corrected for the background to avoid light spurious effects. Reprinted with permission from ref 420. Copyright 2010 American Chemical Society.

fibronectin, and laminin. As a result, successful detection of type I and type III collagens was provided in Stratum papillare and Stratum texticulare skin layers, whereas the other proteins tested gave negative results. As another example, immunofluorescence was applied to the analysis of the cartilage pellicle adhering to 20 000−25 000 years BP fossilized bone excavated from the Enlève cave (Ariège, France), in a way complementary to other analytical techniques such as glycosaminoglycan histochemistry, electron microscopy, mineral phase analysis using X-ray diffraction and energy dispersive spectrometry, amino acid, and hexosamine analyses.433 In particular, following demineralization (0.1 M HCI), cryosections (5 μm) of cartilage pellicle were examined using polyclonal antibovine collagen types I, II, IX, XI rabbit antibodies and antirabbit FITC-labeled IgG. The bovine serum albumin-based mixture was used as blocking agent, and data acquisition was performed using an epifluorescence microscope. As a result, an accurate localization of the target collagens was obtained: type I collagen appeared in the internal layer of the analyzed samples, type II and type XI collagens were detected only in the upper more compact material, and type IX collagen appeared at the edges of some large cavities. Immunofluorescence was also successfully performed on mammoth fossils,403 fibrous cortical and medullary tissues remaining after demineralization of Tyrannosaurus rex bones,148 and in bone fragments and soft tissues from an 80 million year old Campanian hadrosaur, Brachylophosaurus canadensis.411 For example, in a way complementary to ELISA and to other analytical techniques (to study minerals, organic materials, and the sample organization), immunofluorescence using antibodies raised against chicken collagen I was successfully used starting from 0.3 μm EDTA-demineralized cortical and medullary sections of Tyrannosaurus rex bone; the technique had also detected the presence of osteocalcin in the dinosaur tissues.148 The primary antibodies were raised in rabbit (e.g., antichicken collagen I, antiosteocalcin), and the secondary antibodies were biotin-conjugated goat antirabbit immunoglobulin G; fluorophore labeling was provided using Avidin-FITC to produce the biotin−avidin complex.148 The additional evaluation of the antibody reactivity following the collagenase digestion of the dinosaur tissues showed a decreased response. Considering the immunofluorescence analyses performed on the demineralized Brachylophosaurus canadensis extracellular matrix,411 positive reactivities were shown for polyclonal antibodies raised against avian collagen I, osteocalcin, and ostrich whole bone extracts,

serum in phosphate buffered saline. Based on finely polished cross-sections embedded in a poly(methyl methacrylate) resin, the immunofluorescence procedure using hen egg white primary antibody (generated in rabbit) in blocking solution (based on nonfat dry milk) and then antirabbit IgG rhodamine-conjugated secondary antibody (detection using a fluorescence microscope) succeeded in the positive detection of a very thin egg-white layer in a 325 year old sample of polychromy.416 Positive results for the identification of both egg white and milk in painting crosssections based, respectively, on monoclonal ovalbumin and bovine serum albumin recognition were shown; the use of blocking agent was in this study avoided because it might hinder the recognition of an unknown proteinaceous binder.431 Immunofluorescence was also successfully applied to the study of the polychrome binder of Qin Shihuang’s Terracotta Warriors; in particular, the presence of the egg white binder was detected using the corresponding polyclonal antibodies.432 One of the main drawbacks of the technique is related to the unspecific emissions (drawback of all of the fluorescence microscopy-based techniques) such as the emission from the plaster or the gypsum preparation layer of artwork (porous inorganic matrix).420 To reduce the nonspecific background emission, blocking agents can be used during the experiment (as introduced previously in this Review), but also confocal fluorescence microscopy can be used for data acquisition (e.g., monochromatic laser source to scan; very small aperture in the optical path; accurate selection of the wavelength range to be detected).420 To improve the quality of the immunofluorescence images,420 the use of quantum dots was evaluated; QDs are semiconductor nanomaterials. Their long fluorescence lifetimes (several tens nanosecond) offer the possibility of delaying the collection of the immunofluorescence signal (time-gated analysis) after the decay of the background fluorescence (few nanoseconds; as for the common organic dyes). Preliminary promising results were obtained for the detection of ovalbumin in artificially aged egg-based paint samples using time-resolved immuno-fluorescence image (Figure 26). The technique was also proposed for the study of other types of samples such as human mummies.427,428 Starting from frozen sections from air-dried material of 500−1500 year old human mummies from Peru, indirect immunofluorescence was performed using rabbit antibodies directed against extracellular matrix proteins such as collagen and procollagen type I, collagen type II, collagen and procollagen type III, collagen type IV, 37


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Figure 27. In situ immunohistochemistry of demineralized B. canadensis bone matrix and vessels. (A) Incubated with antibodies against avian collagen I. (B) Anticollagen antibodies inhibited with avian collagen, then incubated with tissues as in (A). (C) Demineralized bone matrix digested with collagenase, then incubated with anticollagen antibodies as in (A). (D) B. canadensis demineralized bone matrix incubated with antiosteocalcin polyclonal antibodies (10). (E) B. canadensis vessels incubated with polyclonal antibodies against laminin (10) show weak binding above background levels. (F) Vessels incubated with antibodies against elastin proteins. (G) Vessels digested with elastase, then incubated with elastin antibodies as in (F). (H) B. canadensis vessels incubated with polyclonal antibodies against ostrich hemoglobin. (I) Ostrich hemoglobin antibodies incubated with excess hemoglobin to inhibit binding, then incubated with dinosaur vessels as in (H). Panels (A)−(D) were taken at 79 ms integration, 63× objective; (E)−(I) were taken at 120 ms integration, 63× objective, magnification as shown. Reprinted with permission from ref 411. Copyright 2009 The American Association for the Advancement of Science.

whereas monoclonal antibodies directed against a specific osteocalcin epitope showed no reactivity (single epitope targeted potentially not preserved or not present originally in the studied dinosaur protein) (Figure 27). As mentioned previously, the immunofluorescence technique is regularly associated with complementary techniques to study organic material (e.g., synchrotron radiation-based IR microspectroscopic studies434 to detect the presence of proteins in a studied sample or by MSbased approaches such as TOF-SIMS148 or proteomics411 to identify amino acids or identify accurately a protein/peptide sequence) but also to detect minerals (e.g., X-ray diffraction,433 selected-area electron diffraction148) and morphology (e.g., optical or electron microscopies, field-emission scanning electron microscopy411).

Applied to the study of EDTA-demineralized fossils from mammoth, CL was proposed to detect antigens following a Western blot experiment (proteins separated using SDS-PAGE gel electrophoresis and transferred on a nitrocellulose membrane).403 Antibodies from endogenous antigens of fossils were successfully detected using their respective first antibodies, the horseradish peroxidase conjugated secondary antibody, and a commercially available chemiluminescent substrate. Applied to artwork, CL has the main advantage of not being affected by the autofluorescence of samples (interferences discussed in the previous section).435 For example, applied to ovalbumin immunolocalization in a wood painting dating from the Renaissance (with antichicken egg albumin antibody produced in rabbit, horseradish peroxidase-conjugated polyclonal antirabbit IgG antibody produced in goat, and luminolbased horseradish peroxidase CL detection reagent),435 the successful localization of the target antigen was shown in the 15−30 μm upper section of the sample (the results were confirmed using IR reflectance spectroscopy and fluorescence under UV excitation). The spatial resolution was shown to be at the micrometer scale (i.e., the spatial resolution achieved was able to localize the target protein within the single painting layers, whose thickness is of the order of 5−50 μm),436 and slight or no interferences from the commonly used pigments (smalt, azurite, malachite, hematite, cinnabar, and minium) were observed. The detection limit of the technique, evaluated on ovalbumin spotted on nitrocellulose membrane, was about 0.2 ng/spot, that is, 0.03 ng mm−2. The procedure was further optimized for the detection of animal glues437 and for the simultaneous detection of different proteins such as casein and

4.5. Chemiluminescence for the Study of Ancient Samples

The CL phenomenon is related to the emission of light resulting in a chemical reaction. Luminol is one of the mostly used luminescent compounds, and horseradish peroxidase is often used as a catalyst for the conversion of luminol in the presence of hydrogen peroxide. The technique may involve the use of antibodies via an indirect technique: the target protein is detected using a specific primary antibody, the horseradish peroxidase-labeled secondary antibody detects thus the primary antibody, and the chemiluminescent substrate is added in the experiment for detection using optical microscopy. Applied to the study of ancient samples in the 2000s, the CL technique allowed thus the detection of target proteins in fossils403 or the stratigraphic localization of the target proteins in artwork.435 38


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Figure 28. (a,e) Live images of the cross-sections. (b,f) Chemiluminescence signals corresponding to collagen. (c,g) Chemiluminescence signals corresponding to ovalbumin. (d,h) Overlay between the live images of the cross-sections and the pseudocolored CL images in which the CL signals corresponding to collagen and ovalbumin are represented in shades of blue and red, respectively. Reprinted with permission from ref 437. Copyright 2012 Springer.

ovalbumin436 or collagen and ovalbumin437 in the same crosssection using a multiplexed immunoassay approach. For example, to localize bovine casein and ovalbumin in the same sample, primary antiovalbumin (monoclonal) and anticasein (polyclonal) antibodies were detected using different enzymelabels secondary antibodies (i.e., HRP for the detection of ovalbumin and AP for the detection of bovine casein) and their appropriate chemiluminescent enzyme substrates (i.e., luminol and hydrogen peroxide for HRP and acridan-based chemiluminescent substrate for AP). Before sample incubation, the complex between primary and secondary antibodies was preformed for each antigen independently. Both preformed complexes (against ovalbumin antigen and against casein antigen) were mixed and used for sample incubation; the CL data were obtained using two separate CL detection steps (as previously mentioned, the HRP CL substrate was first introduced for CL detection of HRP-labeled antibody and after sample washing to remove the HRP CL substrate, and CL detection of AP was performed following the addition of the appropriate substrate). Finally, the superimposition of the CL images obtained for each target protein led to the spatial distribution of both proteins in the sample.436 The abilities of multiplexed CL immunoassay were successfully shown during the study of cross-sections of ancient paintings (Figure 28): a wall painting dated from 1773−1774 (sample A) and a painted wood panel of the Renaissance period (sample B).437 Collagen was identified in a thick layer of the wall painting. Considering the wood panel analysis, collagen and ovalbumin were identified in two different layers, respectively, the ground layer and the uppermost layer of the cross-section.

promising results, further developments for the study of more complex samples are currently in progress. Considering the study of proteins in artwork, the use of SERS nanotags complexed to antibodies and detected by Raman spectrometer coupled to optical microscope was successfully reported in 2011.444 The methodology was based on the use of primary antibodies (polyclonal antiovalbumin or antibovine casein) and SERS nanotag-complexed secondary antibody; that is, the secondary antibody was covalently complexed onto the thiol modified nanotag with the sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate cross-linker. Each nanotag had included aggregated-gold spheres (90 nm diameter) coated with a monolayer of reporter molecules encapsulated with a silica shell (30 nm diameter) (the entire particle was ∼120 nm). The dye used in the study was trans-1,2-bis (4-pyridyl)ethylene (spectral profile dissimilar to pigments). This dye has a unique spectral fingerprint when excited by a 785 nm laser allowing spectral identification. Applied to the study of an Italian polychrome sculpture of St. John (ca. 1350−1400),444 the Raman signal for antiovalbumin primary antibody and SERSnanotagged secondary antibody (Figure 29) showed the presence of the egg binder only in the area of the ground layer just below the tin gilding. No Raman signal related to the anticasein primary antibody and the SERS-nanotagged secondary antibody was observed, confirming the lack of casein binder in the sample. The study of other ancient artwork (and models) showed that the immuno-SERS assay is thus able to detect exposed proteinaceous material at the surface of the sample without Raman signal interferences from pigment nor intrinsic fluorescence from the cross-section itself; only cautions related to the formation of agglomerates of SERS-complexed secondary antibody were reported. The SERRS technique was also investigated for the study of artwork.445 In this technique, the Raman reporter is in resonance with the laser excitation wavelength (in SERRS, the analyte has a chromophore close in energy to the excitation frequency used to excite the plasmon and create SERS). In this study, gold nanonarticles (AuNPs) obtained with LASiS were employed for the creation of SERS-nanotags (stable functionalized nanoparticles easily obtained without ligand exchange or extensive purification). The Nile blue A functionalized with lipoic acid, to promote the linking to gold nanoparticles, was used as reporter

4.6. Immuno-Surface-Enhanced Raman Scattering for Painting Analysis and Related Techniques

SERS is a technique based on the enhancement of Raman scattering by organic molecules, with delocalized electron systems adsorbed on rough metal surfaces (e.g., silver, gold, copper). The technique is, for example, applied to the study of the organic colorants of Cultural Heritage samples.438−443 A mobile Raman spectrometer was also developed for acquiring SERS spectra from amino acids (arginine, phenylalanine, methionine) and small peptides (glutathione) adsorbed on silver colloids from model samples.91 On the basis of these first 39


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Figure 29. Raman spectra of immuno-SERS experiments on a cross-section from St. John (MMA 25.120.215). (a) Raman spectrum obtained from an area of the ground layer just below the tin gilding after application of the antiovalbumin primary antibody and SERS nanotagged secondary antibody. The Raman signal for the SERS nanotag indicates the presence of egg binder in this part of the ground layer. The spectrum obtained from the area of the ground layer just below this is shown in (b). The lack of Raman signal for the SERS nanotag in this area implies the absence of egg binder in this part of the ground layer. When anticasein primary antibody and SERS nanotagged secondary antibody were applied to the cross-section in a separate experiment, no Raman signal for the SERS nanotag was obtained in any area of the ground layer (c and d), confirming the lack of casein binder in the sample as was demonstrated by the ELISA screening experiments. Raman spectra (500−1, 800 cm−1) were obtained with a λ0 = 785 nm laser, ∼0.25 mW (25% power), five accumulations per scan. Reprinted with permission from ref 444. Copyright 2010 Springer.

(Nile blue band at 592 cm−1 with the excitation at 785 nm). On the basis of an indirect immunoassay (requiring the use of primary and secondary antibodies) for the detection of ovalbumin in ancient artwork (such as Renaissance painted wood panel), the procedure showed a high resolving power by providing selective location of ovalbumin in the paint sample (19 × 3 μm2 sample; 1 μm step used for analysis). Besides, the immuno-SERS analysis combined with traditional Raman spectroscopy showed other Raman active components, for example, localization of azurite mixed with lead white using the 400 cm−1 CuO stretching band and the 1051 cm−1 symmetric CO32− stretching band, respectively.

4.7. Electrochemical Immunoassay Applied to Artwork

To conclude on immunoassay-based approaches, immunoassay and electrochemical-based detection were recently associated with the detection of proteins in artwork.446,447 A study aiming to detect ovalbumin in a paint cross-section was proposed with the corresponding primary antibody, a secondary antibody labeled with HRP, and a redox couple (i.e., benzoquinone/ hydroquinone). SECM was the technique used to monitor the complex interaction/formation.447 The SECM instrument was equipped with a 10 μm radius platinum ultramicroelectrode (working electrode); an Ag/AgCl electrode was used as a reference electrode, and a platinum wire was the counter electrode. In the first step, benzoquinone was reduced to hydroquinone at the microelectrode. HRP enzyme and H2O2 40


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Figure 30. Cyclic voltammograms recorded before incubation (· · ·); after incubation with extraction solution, 1% BSA, and Anti-IgY-HRP conjugated (0.05 mg mL−1) (− − −); after addition of 1.2 mM H2O2 (−). (A) IU1, (B) RT1, (C) BG1, and (D) OU4. Other conditions are as in Figure 1. Reprinted with permission from ref 446. Copyright 2014 Elsevier.

allowed the hydroquinone oxidation to benzoquinone; the HRP is labeled to the secondary antibody and consequently located in the paint layer due to its interaction with the primary antibody that detects the antigen protein (ovalbumin). The regeneration produced a local concentration gradient and an increment of benzoquinone reduction current that is recorded. The spatial resolution achieved (depending on the geometry and the size of the ultramicroelectrode) is between 5 and 25 μm. Applied to the study of a Renaissance painting, the technique allowed the successful detection of ovalbumin in the external layer of the studied cross-section. An alternative approach aimed to show the presence of the IgY (main serum immunoglobulin in chicken egg yolk; at concentration of 5−10 mg mL−1) resulting from an extract of total proteins of paint samples (using phosphate buffer and sonication; starting material 1 mg). IgY was probed using a system combining (i) a nanoelectrode surface (i.e., NEE) for antigen capture, (ii) the incubation of NEE with the anti-IgY labeled with HRP, and (iii) the detection of HRP using H2O2 as enzyme substrate and methylene blue as redox mediator. The experiment was monitored using a cyclic voltammogram. NEE are prepared through gold deposition within the pores of a polycarbonate membrane. Proteins (including IgY) are expected to bind to the carboxylic groups present on the polycarbonate surface. The membrane characteristics (i.e., pore density and diameter) determine the voltammetric response of electrodes. Electrochemical measurements were performed using a potentiostat equipped with a three-electrodes cell with a platinum counter electrode and an Ag\AgCl (KCl-saturated)

reference electrode (the NEE was used as the working electrode). Methylene blue (redox mediator) is able to transfer electrons from NEE to the HRP-label bound to Anti-IgY. The dotted line cyclic voltammogram (Figure 30) recorded at NEE (before any incubation) shows a reduction peak attributed to the two electron−one proton reduction of methylene blue. The dashed curve cyclic voltammogram pattern (Figure 30) shows the consequence of the treatment of the NEE with IgY, BSA blocking agent, and Anti-IgY-HRP. The full lines represent the analysis results of four samples of wooden icons (XVII−XVIIIth centuries; named IU1 and OU4 in the Figure 30) and two samples of wooden polychrome sculptures (XVI−XVIIth centuries; named RT1 and BG1 in the Figure 30); cyclic voltammograms were recorded following the addition of H2O2. The presence of IgY can be defined by the electrocatalytic current increment (Δicat); that is, Δicat is calculated using experiments with and without H2O2; in particular, values of peak currents Ip and catalytic current icat are used. Figure 30A and B displays the positive detection pattern of IgY; that is, the electrocatalytic current is increased when H2O2 is added to the NEE incubated with the sample extract and anti-IgY-HRP, the reoxidation peak disappeared, and the cyclic voltammogram became sigmoidally shaped (signature of an electrocatalytic process). Figure 30C and D shows a negative response to IgY (the addition of H2O2 does not cause any change, suggesting that IgY and anti-IgY-HRP were not captured). PCA analysis was applied to cyclic voltammogram data resulting in a matrix with 24 samples and 1600 variables (i.e., measured currents at each 41


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Figure 31. MALDI-TOF MS spectrum in reflector mode of the hydrolyzed protein extract from the egg-based painting. Each monoisotopic peak of each detected peptide is used for protein identification by the peptide mass fingerprint method. Reprinted with permission from ref 495. Copyright 2006 American Chemical Society.

fragment ions resulting from the peptide fragmentation.469 Topdown was another analytical way introduced at the end of the 1990s,468,470,471 following works on intact mass measurement of proteins472−474 and first experiments of direct fragmentation of protein cations.475 A top-down experiment involves the measurement of an accurate molecular weight value of an intact protein usually using a high-resolution MS analyzer (e.g., Orbitrap476,477 or Fourier transform ion cyclotron resonance, FT-ICR478,479) and the direct fragmentation of corresponding proteins without preliminary chemical or enzymatic hydrolysis.480,481 The direct fragmentation of proteins can be performed in the source of the mass spectrometer using ISD experiment (radical-induced fragmentation in the MALDI source assisted by the matrix)482 or in the analyzer483 using experiments such as (i) CID or CAD484 (kinetically excited analyte-ions undergo collisions with an inert gas, the translational energy is converted to vibrational energy distributed throughout all covalent bonds, inducing fragmentation when the ion internal energy exceeds the activation barrier required for a particular bond cleavage) and HCD485−487 (based on few higher-energy collisions as compared to CAD that uses hundreds of low energy collisions), (ii) IRMPD488 (fragmentation following the absorption by an analyte-ion of multiple infrared photons), (iii) ECD475,489,490 (exothermic capture of a thermal electron by a protonated analyte causing the analyte backbone fragmentation by a nonergodic process), or (iv) ETD489−491 (electron transferred by an anion to a protonated analyte, inducing fragmentation with analogous pathways to ECD). The analytical strength, the remarkable achievements, and the actual challenges of proteomics-based methods applied to the study of Cultural Heritage samples are detailed in the following sections. In particular, the first sections describe the methodological developments for identification of proteins in artworks, fossils, and archeological materials (museum objects and on-site remains). Beyond protein identification, studies related to the biological species of the identified proteins are presented. These sections include several deeper questions such as the study of the evolutionary relationships among groups of organisms. The last section is related to the actual challenges. The complex question of the study of protein modifications is described. Attempts to

sweep potential). Original variables involved in the characterization of samples containing egg yolk were pointed out, and the discrimination between positive and negative detections of egg yolk was successfully achieved.446

5. PROTEOMIC-BASED METHODOLOGIES AND RELATED MASS SPECTROMETRY-BASED APPROACHES APPLIED TO PALEONTOLOGICAL, ARCHAEOLOGICAL, AND ARTWORK SAMPLES By homology to the term “genome”, the word “proteome” was proposed for the first time in 1995 to designate a protein set expressed by the genome of a cell, a tissue, and an organism at a precise moment of its development and in a precise environment.448−450 The word “proteomics” designates thus the science dedicated to the study of proteins, including their identification, quantification, and the study of their modification (posttranslational and chemical).451−453 Proteomics allows a dynamic description of gene expression. It is mainly based on the use of complementary techniques: (i) gel electrophoresis454−456 and/ or chromatography for analytes purification/enrichment or separation of several hundreds of analytes,457−460 (ii) MS for peptide/protein analysis461−464 since the emergence of MALDI465,466 and ESI467 soft ionization techniques (respectively, crystal solid ionization and liquid electronebulization ionization techniques), and (iii) bioinformatics that integrates genomic or protein databases; it allows the identification of proteins and their modification using MS data. On the basis of these three analytical blocks (separation, mass spectrometric analysis, and database searching), two different strategies for protein identification can be described, bottom-up and topdown approaches.468 Bottom-up is the current proteomics mainstream. It consists of analyzing the peptide mixture resulting from protein hydrolysis (commonly with trypsin, an endoprotease that cleaves proteins at the carboxyl side of Lys or Arg amino acids, except when they are followed by Pro) either by PMF or by peptide sequencing using MS/MS. For PMF, protein identification depends on accurate measurements of peptides molecular weights, whereas for MS/MS, peptides are fragmented in the source or in the analyzer of the mass spectrometer and amino acid sequences are obtained by the elucidation of 42


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Figure 32. Score plot of PC1 and PC2 visually separating the main protein binder classes. Reprinted with permission from ref 504. Copyright 2011 John Wiley and Sons.

using the C18 reverse phase tip was analyzed by MALDI-TOF MS (Figure 31) using a DHB matrix. Using bioinformatic software (MatrixScience), the obtained PMF was compared to the peptide masses predicted from in silico theoretical digestions of the protein sequences available/resulting from the protein/ nucleotide databases created from sequenced genomes or from nucleotidic EST (i.e., short sequence from cDNA sequence), for example, NCBI databanks, SwissProt. The analysis of 200 μg of egg-based models led to the identification of peptides belonging (i) to egg white proteins such as ovotransferrin (∼78 kDa; 12% of egg white proteins) with 17% sequence coverage, ovalbumin (∼43 kDa; 54% of egg white proteins) with 10% sequence coverage, ovomucoid (∼20 kDa; 11% of egg white proteins) with 19% sequence coverage, and (ii) to egg yolk proteins such as vitellogenin II (∼205 kDa; precursor of the major egg yolk protein that contains lipovitellin I, phosvitin, and lipovitellin II) with 7% sequence coverage.492,495 Combined with tandem mass spectrometry experiments (see details in section 5.2), PMF showed for the first time its capability to identify egg white and egg yolk proteins from a few micrograms of paintings dating from the XIVth−XVth centuries.285,495,497 In particular, ovalbumin, lysozyme, ovotransferrin, and ovomucoid egg white proteins were identified in artistic samples, as well as the major egg yolk protein, vitellogenin II (see details in section 5.2). Several alternative protocols referring to sample preparation or analytical procedure were developed and applied to various artwork.498,499 For example, the direct enzymatic hydrolysis of samples using trypsin was proposed without preliminary extraction.493,494 Applied, for instance, to the study of a painting sample dating from the XIXth century (estimated at 0.3 μg), the procedure allowed the detection of 12 peaks attributed to glue, suggesting its use as binder.493 The differences between the standard sample spectra and the paint extract spectra were explained by the potential contamination with other proteins. PMF was also used to study protein additives in ancient mortars.500,501 Applied to the analysis of several samples from the rotunda of Saint Catherine in Znojmo (Czech Republic) dating from the XIIth century, PMF showed the possibility of

explain a particular degradation process or to link protein degradation to aging or particular conditions (e.g., diagenesis, restoration treatments, conservation, pollution) is discussed for different types of ancient samples. Novel methodologies are finally presented with intact protein analysis providing a new level of information. 5.1. Peptide Mass Fingerprint for the Identification of Proteinaceous Binders in Artwork

Proteomics applied to the identification of protein-based materials in artwork samples was mentioned for the first time in the 2000s.285,492−495 The PMF approach is composed of several steps including protein extraction, the controlled hydrolysis of proteins, the MS-based analysis of the resulting peptides, and data processing using bioinformatic tools. The methodology was first developed and optimized on reconstituted egg-paintings composed of lead white, ovalbumin (the main protein of egg white), and linseed oil. The main difficulties of the work were (i) to find a protein extraction solution that extracts proteins from the paint complex matrix without hydrolysis to amino acids and (ii) to optimize the analytical procedure to the study of very low sample amounts (few micrograms). Among all of the evaluated solutions (basic, acidic, proteomics-based buffers including denaturants, reducing agents, and detergents), the best extraction conditions were obtained using sample grinding in an aqueous solution acidified with 1% TFA with a commercial resin (for very thin grinding).285,492 The extracted proteins were analyzed in their native forms using MALDI-TOF to verify the efficiency of the extraction procedure (e.g., the M + H+ ion detected at 44 606 Da was assigned to monocharged ovalbumin). To identify accurately the proteins, a “bottom up” approach was implemented and optimized on model paintings composed of lead white, egg (white and yolk), and linseed oil. Extracted proteins were denatured with guanidine hydrochloride285,492 or trifluoroethanol,496 disulfide bonds were subsequently reduced using dithiothreitol, and finally proteins were alkylated with iodoacetamide. The controlled enzymatic hydrolysis of proteins was performed using trypsin, and the desalted peptide mixture 43


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up to 100 mg was used).506 The mortar from the deeper layer of samples provided spectra with a better signal-to-noise as compared to surface layer samples (comparison of the same MALDI-TOF peaks in spectra from fresh and aged samples). In another framework, PMF was used to study the effect of biorestoration507 based on the activities of enzymes (with or without bacterial cells) on artwork508−510 (biorestoration is one type of cleaning technique among others such as aqueous-based methods, solvent-gel ones, or nanoparticle-based methods510−513). For example, applied to the study of polychrome surfaces, effects of cleaning procedures based on ionic liquids and enzymes (e.g., pepsin that cleaves proteins between hydrophobic/aromatic amino acids, Aspergillus sojae alkaline proteinase that has broad enzymatic specificity) were studied. Experiments were based on the detection of peptides released by enzymatic systems with MALDI-TOF analyses via the classical PMF procedure applied to cotton swabs used for restoration. On the basis of the diversity and abundance of the detected peptides in 900−3200 Da mass range, conclusions were drawn on the best formulation to use for restoration. An analytical protocol for the simultaneous study of proteins and lipids (such as triacylglycerols and phospholipids) from a unique artwork sample was also developed.319,514 The methodology is based on a methanol/chloroform extraction with an ultrasonication step and a centrifugation step, allowing the separation of lipids and proteins. MALDI-TOF analyses were performed, in parallel, on the lipidic fraction and on the peptide fraction resulting from the trypsin hydrolysis of the protein extract. Applied to analyses of several microsamples from the late-XVth century panel painting, the procedure allowed the identification of egg peptides, collagen peptides (explained by the potential use of the animal glue in the gypsum ground layer), and casein peptides (potentially attributed to the decorations), as well as triacylglycerols and phospholipids and their degradation products (suggesting the use of a mixture of drying oil and egg).514 Direct MALDI-TOF analysis of small paint fragments was also proposed for protein and lipid identifications.515 The intact or crushed sample (100 μg) was deposited on a conducting graphite material (colloidal graphite and thermoplastic resin in isopropanol) that was preliminarily deposited on the target. On-target hydrolysis with trypsin was performed. Applied to model egg-based samples, MALDI-TOF spectra showed lysophospholipids and diacylglycerols in the 470−610 m/z range (resulting from the hydrolysis of native phospholipids and triacylglycerols), phospholipids byproducts in the 620−700 m/z range, phospholipids in the 750−820 m/z range, triacylglycerols in the 850−900 m/z range, and peptides from egg yolk vitellogenin-1, vitellogenin-2, vitellogenin-3, and egg white avidin and ovalbumin in the 1000−2000 m/z range. Applied to the study of a polyptych sample dating from 1467, seven peptides from collagen indicated the presence of animal glue; nine peptides from vitellogenin-1 and vitellogenin-2 were also detected, and seven peaks attributed to the lipid fraction confirmed the presence of phospholipids and their degradation products (confirming thus once again the presence of egg). Studies of artwork materials, involving PMF in a complementary way to other techniques (such as optical microscopy, SEM with energy dispersive X-ray spectroscopy, micro-Raman, GC−MS, pyrolysis GC-MS, ELISA, SYPRO Ruby staining), were proposed to fully characterize artwork, including organic but also inorganic materials.23,319,499,516 Focusing on protein compounds, tandem mass spectrometry (see details in the next section) represents a powerful complementary/alternative

distinguishing the main groups of protein additives; that is, three samples showed correspondence with milk proteins, two with collagen-based binder, while proteins were not identified in the two last samples.500 To simplify the data processing and interpretation, the use of lists of the most common peptides that are identified in PMF analyses of various model binders was proposed,499,502 including mixtures and using small sample sizes.499 Characteristic ions for egg yolk, egg white, animal glues, and milk casein were listed. The obtained information was used to propose the first routine application of PMF in a museum laboratory, the Harvard Art Museum in Cambridge.499 In particular, the protocol was adapted to routine application using the following steps: protein extraction and protein denaturation (trifluoroethanol/ammonium bicarbonate and additional ultrasonication), protein disulfide bond reduction and protein alkylation (respectively with tris (2-carboxyethyl) phosphine hydrochloride and iodoacetamide), and trypsin hydrolysis. A cleanup step based on reverse phase chromatography (Zip Tip support) was used to eliminate interferences and improve spectral quality in some particular cases. The method was efficaciously applied to a large number of samples including paintings and museum objects (see details related to the results obtained on museum objects in the dedicated following section), demonstrating the reliability and efficiency of the protocol.499,503 Considering, for example, paintings, analyses of several XIVth century Italian altarpieces in the Harvard Art Museum’s collection (microgram-size samples) showed the successful identification of animal glue binder alone or mixed with whole egg or egg yolk. 499 The results were validated using complementary techniques such as tandem mass spectrometry, GC-MS, FTIR, and ELISA. To distinguish more easily the various proteinaceous binders commonly used in paints (i.e., animal glues, egg white, egg yolk, milk casein), chemometric processing of the data obtained using PMF was investigated by PCA and SIMCA.504 A set of 44 samples including a combination of 10 pigments with four binders was subjected to both chemometric methods. PCA was used to select the most significant peptide peaks in MALDI-TOF spectra for binder discrimination, and SIMCA was used to create a supervised pattern recognition model (based on the proximity of unknowns to different groups in the training set). The discrimination of egg white, egg yolk, milk casein, and glues was successfully achieved (Figure 32), and in the case of glues, sturgeon glue could be differentiated from mammalian glues. The procedure was applied to several samples from a XVIth century altarpiece. Despite the low signal-to-noise ratio obtained on MALDI-TOF spectra and thus the slightly biased chemometrics data as compared to standard samples (attributed to the paint natural aging), positive identifications of mammalian glue were obtained. PCA based on MALDI-TOF data was also used to better understand the degradation process of collagen-based binders due to UV exposure (up to 3000 h of UV irradiation) and the interactions with pigments.505 In particular, the PCA plots related to pure rabbit glue mixed with one inorganic pigment, that is, cinnabar or azurite, with and without aging, showed different profiles suggesting different aging behavior depending on the nature of the pigment. The formation of protein−copper complex was suggested to justify the additional photostabilization of glue tempera in samples containing azurite. Further FTIR spectra showed conformational changes in the secondary structure of collagen during photoaging experiments. The effect of natural aging (up to 5 years: 1 year outdoor and 4 years indoor) was also studied on model mortar samples (5 mg 44


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Figure 33. MS/MS spectrum of the triply charged ion at m/z 551.61, from the hydrolyzed extract of Benedetto Bonfigli’s triptych, presenting the y and b fragments of the ovotransferrin peptide 443−457 (TDERPASYFAVAVAR). F* and Y* correspond, respectively, to the phenylalanine and tyrosine immonium ions. Reprinted with permission from ref 495. Copyright 2006 American Chemical Society.

with good signal-to-noise ratios and high sequence coverages (using y+, y2+, b+, and b2+ fragment ions) were generated, allowing the accurate identification of several egg yolk and egg white proteins based on several accurate peptide sequences (e.g., MS/MS spectrum of an identified peptide of the ovotransferrin shown in Figure 33).495 The results showed that the two Renaissance paintings were prepared with a more complex formulation than a “tempera” (containing only egg yolk), because it was a mixture of egg white, yolk, and oil, often called “tempera grassa” (known to produce particular chromatic effects such as bright and shimmering effects). Several alternative protocols or analytical procedures were subsequently proposed.517−520 For example, paint samples were suspended in ammonium bicarbonate buffer and submitted to ultrasonic bath (followed by a classical dithiothreitol reduction, an iodoacetamide alkylation, and a trypsin-based enzymatic hydrolysis step);517 the demonstration of the protocol efficiency was shown by the study of wall painting samples dating from the early twentieth century (0.1 cm2 surface size) that have resulted in the identification of egg white proteins (lysozyme, ovalbumin, ovotransferrin), egg yolk proteins (vitellogenin), and caseins (αS1 and β-caseins). Microwave-assisted digestion was also reported (15 min digestion at 700 W instead of several hours for classical procedure).518 Analyses carried out on model paintings (based on hematite or malachite and egg, milk, or animal glue), treated or not with Paraloid B72 (an acrylic resin used to consolidate the fragments), have successfully resulted in protein identification. However, applied to fragments recovered by the collapsed vaults of the Basilica of St. Francis of Assisi, protein digestion without pretreatment showed difficulties for protein identification. The trypsin-hydrolysis incubation time was also evaluated.501 Model mortar samples containing bovine milk, curd, and whey were directly placed into the tryspin-based solution, and 2 h digestion was found to be the optimal duration (the selection of the best duration was based on the greatest number of peptide peaks observed in spectra). With this protocol, MS/MS experiments led to the identification of several milk proteins such as α-lactalbumin, β-lactoglobulin, α-casein, and κ-casein (starting materials: 5 mg of fresh sample, 50 mg of 9

technique to PMF as it allows a deep structural approach with the accurate identification of the peptide backbone sequence and its modifications. Thus, protein identification can be accurately performed on the basis of a few peptidic sequences. Additional information can also be obtained such as the biological origin of the proteins or information on sample degradation state or aging (see details in the next sections). 5.2. Tandem Mass Spectrometry Applied to Artwork Samples

The study of proteins in artwork using tandem mass spectrometry was proposed for the first time in the 2000s.285,495,497 On the basis of peptide fragmentation in the MS instrument (commonly in the analyzer using CID/CAD experiments), MS/MS allows the characterization of exact amino acid sequences of peptides, providing thus the accurate identification of proteins.469 It allows also the identification of protein modifications. The sample preparation is similar to PMF; however, the analytical workflow is modified (e.g., using different MS instruments, using different MS acquisition mode, integrating supplementary analytical steps such as nanoLC separation of analytes before data acquisition). The first application of the MS/MS methodology was based on sample preparation using the following steps:285,492,497 protein extraction was performed using sample grinding in 1% TFA solution, and proteins were denaturated (guanidine hydrochloride495 or trifluoroethanol496), reduced (dithiothreitol), alkylated (iodoacetamide), and enzymatically hydrolyzed (trypsin). The analytical procedure using nanoliquid chromatography (nanoLC) online with nanoESI-Qq-TOF MS/MS analysis was optimized for very low amounts of samples. In particular, the analysis was optimized for a 1 μL-sample volume, and the nanoLC was equipped with a C18 precolumn (0.5 cm × 300 μm) for sample desalting/concentration and a C18 nanocolumn (15 cm × 75 μm) for analyte separation at 200 nL/min solvent flow for 3 h gradient online with MS and MS/MS analysis. Applied to the study of 10 μg samples dating from the Renaissance, that is, the Benedetto Bonfigli triptych “The Virgin and Child, St. John the Baptist, St. Sebastian” and the Niccolo di Pietro Gerini painting “The Virgin and Child”, MS/MS spectra 45


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Table 5. Species-Specific Peptides of Collagen Proteins Identified for the Gilt Sample Analyzed Entirely and the Different Layers Constituting This Sample Analyzed Separatelya identified proteins collagen alpha1(I) chain precursor

collagen alpha2(I) chain precursor

collagen alpha1(III) chain

taxonomies

protein ID nos.

specific peptides

positions

A

Bos taurus (bovine)

P02453 (CO1A1_BOVIN)

GARGEPGPAGLPGPPGER GEPGPAGLPGPPGER GETGPAGRPGEVGPPGPPGPAGEKGAPGADGPAGAPGTPGPQGIAGQR GAPGADGPAGAPGTPGPQGTAGQR GPAGAPGTPGPQGIAGQR GLVGEPGPAGSKGESGNKGEPGAVGQPGPPGPSGEEGKR GSTGEIGPAGPPGPPGLR GEQGPAGPPGFQGLPGPAGTAGEAGKPGER GIPGEFGLPGPAGAR GPSGPPGPDGNKGEPGVVGAPGTAGPSGPSGLPGER SGETGASGPPGFVGEK PQGLLGAPGFLGLPGSR GYPGNAGPVGAAGAPGPQGPVGPVGK GEPGPAGAVGPAGAVGPR IGQPGAVGPAGIR IGQPGAVGPAGIRG GAPGPQGPPGAPGPLGIAGLTGAR

469−486 472−486 910−957 934−957 940−957 341−379 380−397 542−571 572−586 608−643 829−844 861−877 947−972 977−994 1066−1078 1066−1079 777−800

× × × ×

Bos taurus (bovine)

Bos taurus (bovine)



× × × × ×

B

C

× × × × ×

× ×

× × × ×

× × × × × ×

× ×

a

Samples: (A) gilt sample; (b) plaster support; (C) upper layer (gilding bole and gold lsaf). Experimental details are presented in the Supporting Information of ref 496 (Tables S-6−S-8). Reprinted with permission from ref 496. Copyright 2011 American Chemical Society.

wall painting of the first century AD from an Italian archeological site.288 Considering the study of the biological origin of glues used in artwork, additional difficulties have to be considered: (i) collagens are high molecular weight proteins (150−400 kDa) resulting in complex mass spectra (often without full sequence coverage), and thus an adapted analytical methodology has to be developed to obtain the most informative spectra (e.g., adapted nanochromatography separation online with MS); and (ii) there is a lack of sequenced collagen proteins for several species, proteomics is dependent on the databases that are in constant evolution; however, in the case of the analysis of a non sequenced species, de novo sequencing (sequencing without prior knowledge of the amino acid sequence) can be performed on the basis of MS/MS data to obtain accurate information on peptide backbones (similarities or dissimilarities as compared to the existing proteins in databanks). An attempt to assign the protein origin of the collagen identified in samples dating from the XVth to XVIIth centuries was proposed without the use of an alignment program.519 On the basis of the peptides identified using the SwissProt database, manual discrimination was performed (presence of sequences in several species or not), and hypothesis was provided on the bovine origin of the glue using two peptides. The accurate identification of the biological origin of glue was successfully shown using a methodological approach based on online nanochromatography coupled to high resolution mass spectrometry (the hundreds of m/z collagen peptide peaks detected could be highly resolved) and a homemade bioinformatic tool for sequence alignment (homemade parser based on BioPython integrating the lastest NCBInr database).22,496 The discrimination of bovine, rabbit, and fish glues was successfully achieved starting from a few micrograms of a commercial glue, a 100 years-old glue from the “Totin Frères”, and a gilt sample dated from the XVIIIth century (∼50 μg).496 Using the sample preparation procedure described previously, the analyses of ancient gilt sample have resulted in the identification of collagens α1 type I, type II, and type III, and collagen α2 type I. For example, considering collagen α1 type I, a

months-aged sample). The effects of sample pretreatment and the cleanup strategy before the MS-based analysis were also investigated and optimized519 during the study of models formulated with different binders (rabbit skin glue, egg white, egg yolk, and casein) and pigments (azurite, lead white, red earth, chalk, chrome yellow, lead tin yellow, verdigris, carbon black, vermillion, Prussian blue). In particular, denaturing agents (urea, thiourea) and a detergent (sodium dodecyl sulfate) were used with short sonication cycles to dissolve and denaturate the proteins before the reduction (dithiothreitol), the alkylation (iodoacetamide), and the enzymatic hydrolysis steps. Following the sample treatments, the resulting peptide mixtures were purified before analyses, and three different chromatographic phases were evaluated (HLB, two SCX, HILIC). The best cleanup results (sequence coverage criteria) were obtained using HILIC chromatography. Applied to the analysis of artwork samples dating from the XVth to XVIIth centuries, the successful identification of peptides belonging to collagen proteins was shown, indicating the use of glue as binders. Complementarily to the accurate identification of the protein material, tandem mass spectrometry with the support of bioinformatic tools was used to determine the biological origin of proteins in artwork.22,288,496,517,519 This information remains of major interest to understand a painting formulation, an artistic technique, or to propose restoration campaigns respecting the artist’s technique and used materials. Applied to the discrimination of milk origin, studies of the peptides from αS1-casein (23 kDa) that are species specific (e.g., bovine, sheep, goat) were investigated.517 In particular, using a dedicated software (i.e., Clustal Omega provided by the EMBL-EBI), the alignment of casein peptide sequences obtained from the MS/MS data and those available in the public databases for different animal species was performed. As a result, it was shown that sheep milk was used as binder (two discriminant peptide sequences); several peptides were also found to clearly discriminate sheep and goat from bovine species. A similar analytical approach was used to identify buffalo origin of the αS1-casein present in the 46


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sequence coverage of 41% was achieved using 28 peptides (results from the total sample analysis without layer separation). Further data processing based on sequence alignments showed that 4 peptides among these 28 were specific of the bovine species (Table 5). Eight additional peptides specific of the bovine species were identified for collagen α2 type I and one for bovine collagen α1 type III (no specific peptides identified for collagen α1 type II). The case study of fish glue remains particularly difficult because very few sequences are available in databases. For example, the collagen from sturgeon glue, often used in the artistic field, is not yet sequenced (UniProt Knowledgebase release 2013_08); only five partial sequences (19−72 kDa) are described for Adriatic and Siberian sturgeons (respectively, Acipenser naccarii and Acipenser baerii). Using the currently available databases, the fish species used in glue manufacturing cannot be identified with accuracy, but the discrimination between fish glue and glues from other origin (such as bovine glue or rabbit glue) still remains possible. For example, applied on a complex multilayered polychromy dating from the XVIIth century, a thin fish glue layer located between the silver leaf and the glaze of the polychromy was identified for the first time (localization using the SYPRO Ruby staining, see Figure 1, and the identification using MS/MS and sequence alignment processing).22 The fish origin was shown using discriminant sequences with homologies to other sequenced fish species; 7 peptides showed sequence homologies to 14 fish species. It can be noted that glue binders of two different origins were identified in the same sample, from fish and bovine origins (respectively present in the thin layer covering the silver leaf of the polychromy and in the ground layer).22 To discriminate fish species, due to the lack of databases (as previously mentioned) the study of models is required. For example, anchovy could be discriminated from bonito on the basis of several peptide markers showing one or several amino acids substitutions.521 Thus, to improve the identification of the biological species of collagen, a library of MS/MS spectra obtained from reference glues from various species was developed.522 In particular, reference samples with a particular interest in conservation were studied such as rabbit (Oryctolagus cuniculus), bovine (Bos taurus), Siberian sturgeon (Acipenser baerii), pig (Sus scrofa), deer (Cervidae family), goat (Capra aegagrus), hare (Lepus europaeus), carp (Cyprinidae family), and trout (Salmoninae subfamily of the family Salmonidae). The results obtained with MS/MS experiments, shown in Table 6, have integrated spectra that have, at least, three different matches and belong to at least two different glue samples (not necessarily of the same species) (excluding thus peptides from keratins resulting often from sample handling or other contaminations). This database was successfully applied during studies of artwork dating from the XVIIth and XIXth centuries; experiments have resulted in the identification of rabbit and fish glues (probably Siberian sturgeon or close species in evolution). Recently, the bottom up proteomics based on MS/MS showed its capability to identify bovine collagen markers used as adhesive on a bone sculptureinlaid wooden artifact from the Xiaohe Cemetery in Xinjiang in China.523 Two specific peptides of bovine origin were identified from α1 type I and α2 type I collagens (10 mg of starting archeological material). Another recent example had pointed out the successful identification of collagen peptides using proteomics (in parallel with other techniques) in a 180−200 AD Romano-Egyptian panel; in particular, the collagen α1 type III was identified suggesting the use of skin-based glue such as cow hide.421

Table 6. Summary of the Mascot/SwissProt Search Results with Total (T) Number and Number of Unique (U) Identified Peptides for Each Proteina protein chain bovine hide glue 3359 collagen type I alpha-1 (COL1A1)

collagen type I alpha-2 (COL1A2) collagen type III alpha-1 (COL3A1) rabbit skin glue 581 collagen type I alpha-1 (COL1A1) collagen type I alpha-2 (COL1A2)

collagen type II alpha-1 (COL2A1) collagen type III alpha-1 (COL3A1) sturgeon bladder glue 3362 collagen type I alpha-1 (COL1A1)

collagen type II alpha-1 (COL2A1) a

species

T

U

Bos taurus (cattle) Mus musculus (mouse) Gallus gallus (chicken) Mammut americanum (American mastodon) Bos taurus (cattle) Homo sapiens (human) Gallus gallus (chicken) Bos taurus (cattle) Rattus norvegicus (rat)

9 4 2 3

4 1 1 2

8 5 4 5 2

1 1 2 4 1

Mus musculus (mouse) Canis familiariz (dog) Homo sapiens (human) Bos taurus (cattle) Canis familiariz (dog) Oryctolagus cuniculus (rabbit) fragment Homo sapiens (human) Mus musculus (mouse) Rattus norvegicus (rat) Mus musculus (mouse)

9 8 8 3 3 2

1 1 1 1 1 1

2 2 2 2

0 0 0 2

Rattus norvegicus (rat) Mus musculus (mouse) Bos taurus (cattle) Homo sapiens (human) Canis familiariz (dog) Xenopus laevis (African clawed frog) Cynops pyrrhogaster (Japanese fire belly newt)

4 4 4 3 3 3

1 1 2 1 1 2

5

5

Reprinted with permission from ref 522. Copyright 2012 Elsevier.

5.3. Proteomics in Archeology and Paleontology

5.3.1. Identification and Sequencing of Proteins from Fossils and Ancient Bones (Paleoproteomics). The study of proteins in fossils using PMF and peptide sequencing experiments was proposed in the 2000s.524−527 The first experiments focused on osteocalcin protein due to its high affinity for the hydroxyapatite mineral phase of bones (and thus its potential preservation over time),178 and because it is the second most abundant bone protein (1−2% of the total).178 It can be pointed out that this protein was positively detected in ancient fossils using techniques such as immuno-based detection.403,408,409 In this case, based on EDTA or HCl demineralized bones from bovine, bison, elk, mastodon, and walrus dating from the present to 450 000 years BP, MALDITOF mass spectrometry was employed as a complementary technique to SDS-PAGE gel electrophoresis (silver staining) and radioimmunoassay (polyclonal rabbit antisera against pure bovine osteocalcin and 125I tracer) to study osteocalcin.524 In particular, the employed techniques showed positive detection of the target protein partially purified using reversed phase high performance liquid chromatography (5 g of starting material). For example, the decarboxylated form of osteocalcin was detected (m/z 5722) using MALDI-TOF MS, and concen47


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Figure 34. Postsource decay (PSD) of tryptic peptide 1−19 from older than 55.6 ka fossil bison osteocalcin. Precursor ion, MH+, is represented by peak at the highest m/z (2066). Metastable decay products from cleavage of peptide backbone were labeled using Roepstorrf nomenclature (Roepstorff and Fohlman, 1984). B. priscus sequence appears above mass spectra. For comparison, modern cow is Tyr-Leu-Asp-His-Trp-Leu-Gly-Ala-Hyp-Ala-Pro-TyrPro-Asp-Pro-Leu-Glu-Pro-Lys (GenBank GEBO gi: 538590). Two sequences differ by 129 Da (Gly vs Trp at position 5). Reprinted with permission from ref 525. Copyright 2002 Geological Society of America.

osteocalcin sequences generated for chimpanzee (Pan troglodytes) and gorilla (Gorilla gorilla gorilla) and a partial sequence for orangutan (Pongo pygmaeus) allowed the identification of amino acids modifications as well as the posttranslational hydroxylation that was present or not in the studied species. Gorilla showed Hyp in the ninth position (in a mixture with Pro in the ninth position) of the osteocalcin sequence, whereas Neanderthals, humans, chimpanzee, and orangutan included the Pro at this same position. From this observation, conclusions related to the vitamin C diet of the studied species were drawn, due to the fact that vitamin C enters in the hydroxylation mechanisms based on the action of an enzyme (prolyl-4hydroxylase) and the presence of a consensus sequence (LeuGly-Ala-Pro9-Ala-Pro-Tyr present in most mammals).526 Equid bones dating from 42 000 years were also studied using PSD, CID, and Edman degradation.527 The Edman degradation technique528 consists of a cyclic degradation of peptides based on the reaction of PITC with the free amino group of the Nterminal residue such that amino acids are removed one at a time and identified as their phenylthiohydantoin derivates. Ancient sequences were compared to sequences of modern horse (Equus caballus), zebra (Equus grevyi), and donkey (Equus asinus). Similar sequences were obtained for studied samples (with all of the techniques used); however, particular modifications such as hydroxylation and deamidation were observed, for example, deamidation of Gln to Glu (position 39). The deamidation reaction occurs during fossilization and aging (see details in the section related to chemical modifications analysis, where deamidation is described and is known to occur during aging). The survival of osteocalcin in various archeological bones was investigated using protein analysis and mitochondrial DNA analysis. The results indicated that osteocalcin is highly sensitive to degradation (e.g., temperature of the archeological site).529

trations of osteocalcin in the studied fossils were measured from 0.2 to 450 ng/mg bone using radioimmunoassay. A peptide sequencing approach was also successfully implemented based on osteocalcin enzymatic digestion (with trypsin) and PSD experiments (matrix used: α-cyano-4-hydroxycinnamic acid). PSD is a laser-induced fragmentation process: when ions have acquired a sufficient internal energy during the MALDI desorption process, they leave the source, fragment in the field-free section of the instrument, and fragment ions are separated as a function of their kinetic energy by the time dispersions induced by the electrostatic reflector. The analyzed peptides were derivatized with tris-trimethoxyphenyl phosphonium acetyl N-hydroxy succinamide ester to form the N-terminal tris-trimethoxyphenyl phosphonium acetates of tryptic peptides (TMPP acetyl-derivatives).524 On the basis of a similar analytical procedure (without TMPP derivatization), PSD experiments have provided the first complete osteocalcin sequences of two bison bones (Bison priscus) dating from 55.6 and 58.9 thousand years (Figure 34).525 Further peptide mass fingerprint experiments showed similar profiles for ancient and modern bison osteocalcins (starting material 20 mg), indicating the absence of diagenetic products. However, a difference in a peptide sequence could be shown using PSD experiments (Figure 34; matrix used α-cyano-4-hydroxycinnamic acid or 6-aza-2-thiothymine); that is, the first peptide residue (1−19) differed by an amino acid substitution between cow and bison species (respectively Trp versus Gly). This observation showed that protein sequences, in a way complementary to DNA, can be used to investigate molecular phylogenies over a large time period. Protein sequences of fossil hominids from two Neanderthals dating from 75 000 were also successfully analyzed using CID experiments.526 It was shown that the Neanderthals have a osteocalcin amino acid sequence identical to that of modern humans. Additional experiments based on the complete 48


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Figure 35. Reflectron MALDI-TOF mass spectra, obtained on digestion of mastodon and woolly mammoth collagen with trypsin and elution into two fractions (10% and 50% acetonitrile in 0.1% TFA). Reprinted with permission from ref 542. Copyright 2011 Elsevier.

acid sequence variation) during the study of 36 modern species resulting in a phylogenetic analysis from sequencing experiments, published data, and genome-derived sequences.539 Further investigations have identified 92 marker peptides during the study of 32 different modern animal species.540 Thus, the possibility of recovering evolutionarily significant molecular information from fossils was investigated using collagen-based proteomics (ZooMS).540 On the basis of a similar analytical approach, animal species of archeological bones from excavations dated from late Neolithic site were successfully discriminated on the basis of a unique peptide of collagen showing two amino acid substitutions (the peptide was isolated using C18 reverse phase fractionation); that is, sheep species was differentiated from goat (starting material 2 mg of acid-insoluble collagen).541 Further investigation of the collagen survival and its use for species identification were studied using proteomics experiments but also using simulation models based on the thermal age (chemical age and thermal history of the site).536 For example, a systematic study of collagen PMF and peptidic sequences was successfully performed starting from a large range of samples of various species (including the taxonomic orders Carnivora, Artiodactyla, and Cetacea) and from various ages dating back approximately 1.5 million years. Another example is the phylogenetic study of collagen sequences of mammoth and mastodon bones.542 On the basis of PMF and MS/MS

Thus, the study of collagen was recommended; collagen type I is the major protein in bones. Multitechnical and multimethodological investigations were provided to study bone diagenesis and the related degradation of proteins such as collagen.178,182,530−533 Among these investigations, mass spectrometry-based studies proved that collagen may survive in ancient samples using the analysis of its constitutive peptides.534,535 For example, collagen peptides were successfully identified in bones from Holocene and Pleistocene periods dating back ∼1.5 million years,536 in samples from a 68 million year old dinosaur,537 and in hadrosaur samples from an 80 million year old.411 The main steps of analytical procedures were based on sample demineralization (e.g., HCl,536 EDTA411) or sample treatment with ammonium bicarbonate (partial extraction but bone artifacts undamaged post-treatment),538 enzymatic protein hydrolysis (most commonly with trypsin, but the use of other enzymes such as type III bacterial collagenase that cleaves repeating sequences such as the GlyPro-Hyp sequence of collagen was also described539), peptide purification (commonly with reverse phase chromatography), and PMF analysis and peptide sequencing (using CID experiments). Beyond the identification of collagen peptides, the possible identification of biological species with marker peptides was also shown. For example, collagen (I) α2 chain carboxytelopeptide was used as a biomarker of species (amino 49


2878.421

1458.685

1571.769

1531.738

1161.589

1305.631

1609.734

1577.782

960.487

730.740

786.901

766.877

582.160

653.824

805.875

789.898

0.0005

0.0012

0.0013

0.7164

0.0005

Orbitrap

Orbitrap

Orbitrap

ion trap

Orbitrap

Orbitrap

0.0180b

Orbitrap

ion trap

b

instrument rank

0.7793

0.0047

mass error

1

1

1

1

1

1

1

1

54.3

40.5

56.9

65.8

52.7

37.2

73.7

40.0

mascot score

0.023

0.54

0.012

0.0015

0.023

0.84

0.00027

0.59

mascot expectation value

3.97

2.32

2.63

2.48

2.70

3.13

3.99

4.53

sequest Xcorr

search stats; synthetic peptide search stats



search stats; ostrich peptide search stats

validation

GLPGESGAVGPAGPP(OH)GSR

GSN(deam)GEP(OH)GSAGPP(OH)GPAGLR

GPSGPQGPSGAP(OH)GPK

GVQGPP(OH)GPQGPR

GETGPAGPAGPP(OH)GPAGAR

GATGAP(OH)GIAGAP(OH)GFP(OH)GAR

GLTGPIGPP(OH) GPAGAP(OH)GDKGEAGPSGPPGPTGAR GSAGPP(OH)GATGFP(OH)GAAGR

peptide sequence collagen α1(1) collagen α1(1) collagen α1(1) collagen α1(1) collagen α1(1) collagen α1(1) collagen α2(1) collagen α2(1)

protein

T. rex

T. rex, chicken, alligator, and opossum chicken, alligator, rat, and opossum chicken and alligator

T. rex, chicken, alligator, and amphibia chicken

T. rex, chicken, and mammals

ostrich and mammals

BLAST sequence identity

a m/z values of the peptide ion, molecular weight, mass spectrometer used, database search engine scores, expectation values, sequence validation method, and sequence identity based on BLAST searches versus the all-species NCBInr protein database and internally acquired ostrich and alligator sequences are shown. The interpretation of the sequence GLPGESGAVGPAGPP(OH)GSR was aided by the high mass accuracy of the Orbitrap, because hydroxyproline is more accurate than isoleucine/leucine at position 15 by 0.0364 Da. Reprinted with permission from ref 411. Copyright 2009 The American Association for the Advancement of Science. bFor two sequences [GLTGPIGPP(OH)GPAGAP(OH)GDKGEAGPSGPPGPTGAR and GATGAP(OH)GIAGAP(OH)GFP(OH)GAR] acquired with the Orbitrap, MS/MS was triggered on the m/z ratio representing the 13C stable isotope containing ion rather than the monoisotopic version.

Mr (calcd)

m/z (obsd)

Table 7. Collagen α1(I) and α2(I) Sequences Acquired by Ion Trap and Orbitrap Mass Spectrometry for B. canadensisa

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Figure 36. Human STRING network of mammoth orthologous proteins. Blue lines between nodes represent functional associations between proteins; the thickness of the lines represents the level of confidence in the association reported. Reprinted with permission from ref 555. Copyright 2012 American Chemical Society.

experiments, the discrimination between mammoth and mastodon species was pointed out (e.g., PMF showed in Figure 35); the study has thus provided the estimated relationships and divergences between elephantids and mastodon (from Oligocene to Pliocene-Pleistocene) with the amino acid substitutions identified. The possibility of studying collagen extracted from ancient teeth (e.g., 8500 year old human dental pulp,543 dentine from Neolithic samples544) was also shown using PMF; for example, collagen-PMF was used for taxonomic identification in various faunal remains dating from the Neolithic period.544 Fish bones from modern and archeological samples were also analyzed through their collagen fingerprints.545 The study included several sample treatments before the protein enzymatic hydrolysis (trypsin) and the resulting peptide analyses; for example, the modern fishes were boiled, baked, or soaked in cool water to remove tissues. Both HCl-based demineralization and warm ammonium bicarbonate were evaluated to remove the proteins from the bone matrix (few bones needed for analysis). Four orders of modern fishes were analyzed (Clupeiformes, Salmoniformes, Gadiformes, and Perciformes), among which were eight species (herring, salmon, trout, cod, haddock, hake, bass, and sand eel). The archeological samples included the same

species as the modern selection (cod, haddock, herring, and bass) but also related species (whiting and mackerel) and distantly related species (ray and plaice) with several bone samples morphologically unidentifiable. Analyses have provided clear differences between the studied species using discriminant peptides and statistical methods (such as partial least-squares linear discriminant analysis). In particular, 89 biomarkers were successfully identified. It can be pointed out, for example, that one of the unknown specimens could be classified confidently (haddock) with some other archeological and modern specimens. However, the number of species in the database represents the main limitation of the approach; the database will be thus extended using additional references samples to improve the classification abilities and the confidence levels. Recently, collagen fingerprints were also used to identify cetaceans and pinnipeds marine mammals.546 The discrimination of various archeological specimens from sites ranging from the Mesolithic until the Early Modern period was successfully achieved.546 Mass spectrometry-based PMF and sequencing have shown evidence for the survival of protein compounds in fossil materials older than 1 million-years-old, providing thus new insights in the 51


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fields of evolutionary biology.535,547 Collagen survival was shown during analyses of fossils of Tyrannosaurus rex537,548 dating back 68 million years and fossils of mastodons dating back 160 000− 600 000 years.537 The sequenced peptides were used to compute a phylogenetic tree using sequence alignments (with BLAST alignment bioinformatic tool), resulting in high similarities between the Tyrannosaurus rex collagen peptides and those of birds (Gallus gallus) and between mastodon (Mammut americanum) and other mammals, including elephant (Loxodonta africana).549 Data interpretations and statistical analyses were discussed 550−553 showing the importance of the precautions taken against potential contaminants during excavations and during sample analyses (e.g., analyses by more than one mass spectrometry laboratory). Thus, an investigation using a multitechnical approach (microstructural and immunological-based techniques used) and special precautions during collection and conservation of samples (e.g., use of sterile instruments, samples wrapped in layers of foil, placed in sealed jars with desiccation crystals) was proposed to study bone fragments and soft tissues from 80 million year old Campanian Hadrosaur (Brachylophosaurus canadensis).411 In particular, sequences from α1 and α2 type I collagens were successfully provided (Table 7), including high precautions for sequences validations (e.g., scoring statistics, decoy databases, manual inspection, spectral comparisons to synthetic peptides spectra, use of an algorithm from the NIST including database of 200 000 random peptide fragmentation spectra from various taxa). As a result, the obtained data on the B. canadensis have supported the bird-dinosaur clade. Protein survival in fossil bones from dinosaur was further undertaken using simulations related to the localization of dinosaur peptides (from T. rex and B. canadensis studies) in the collagen fibril structure.554 Molecular models of the vertebrate collagen fibril were derived from extant taxa such as human and rat microfibrils. In particular, simulations have shown that the studied dinosaur peptides were not localized in the most exposed parts of the collagen fibril (explaining potentially the preservation of peptides). Additionally, four peptides (among the 11 studied peptides) were shown to be potentially localized in a collagen key region for cell−collagen interactions and tissue development. Beyond the studies of osteocalcin and collagen in fossils, a proteomic approach allowed the successful identification of 126 proteins from a 43 000 year old Siberian mammoth femur (Mammuthus primigenius) preserved in permafrost (starting material ∼75 mg; EDTA-based demineralization).555 In particular, plasma proteins (e.g., apolipoprotein A-IV, albumin, plasminogen) were identified as well as membrane and intracellular proteins (e.g., moesin, myosin), cytoskeletal keratins, collagens and other extra cellular matrix bone, calcium, and fibril related proteins (e.g., alpha-2-HS-glycoprotein, osteopontin). The identified sequences showed several amino acids substitutions; for example, two amino acid substitutions in albumin sequence were identified for mammoth species in comparison to African and Indian elephant sequences. A consistent subset of the identified proteome was also identified in two other samples of more recent mammoth bones (19 000 years) preserved in a temperate environment. Functional networks formed by the interacting proteins within the observed proteome were searched using a dedicated algorithm (STRING). As a result, 99 proteins were functionally associated (Figure 36) with at least one other putative mammoth orthologue (originated by vertical descent from a common ancestor gene), and further data processing related to

clusterization showed that one cluster could be associated with bone extracellular matrix. Protein survival in bones was also studied using a set of bovine bone samples ranging in age from approximately 4000 to 1.5 million years old (and bovine specimens from 10 000 years old in a warmer climate (Cyprus) for thermal history comparison).556 The proteomics methodology included the removal of collagen using bacterial collagenase. Among the non collagenous proteins that were more easily recovered in ancient samples were serum proteins such as albumin and alpha-2-HS-glycoprotein. Extracellular matrix proteins associated with collagen, such as biglycan, were also identified in ancient bones. In particular, the alpha-2-HSglycoprotein appeared as an interesting target for the species identification and phylogenetic studies. Proteomics, in a way complementary to DNA analysis149,557 and other techniques (such as immunology), appears thus as an essential analytical tool to study ancient protein sequences from fossils.534,558 Beyond the phylogenetic studies (suggesting new insights into the evolutionary history411,542,559), it can be pointed out that proteomics offers various additional information on ancient samples. The following examples show the level of information currently obtained on ancient proteins. One illustration is the identification of bacterial highly antigenic virulence proteins and inflammatory and anti-inflammatory host proteins from human medieval dental calculus.560 Another example is the detection of protein biomarkers of osteogenic sarcoma (e.g., annexin A10, transferrin, BCL-2-like protein) in bones dating back 2000 years,561 or the detection of proteins from the immune system response (e.g., serine protease inhibitor, transthyretin) of a 500 year old Inca mummy.562 5.3.2. Identification of Proteins and Protein Residues in Archeological Samples. In parallel with the study of proteins in fossils, proteomics methodology was also adapted to the study of a broad range of archeological samples (e.g., remains in ancient potsherds, textiles, egg shells, mummy skin) for a better comprehension of object use or human tradition. The proteomics protocol (mainly the protein extraction procedure) differs in function of the sample nature, for example, proteins trapped in a matrix such as ceramic versus proteins constitutive of a sample such as skin or textile fibers. The application of proteomics to an archeological material was described for the first time at the end of the 2000s,563 during the study of protein remains in an ancient potsherd fragment. It can be pointed out that the study of protein remains in an archeological shard is challenging because proteins can be easily leached during burial due to their hydrophilic properties (as compared to lipids, for example). The study aimed to identify protein remains in an Iñupiat potsherd fragment (possibly 1200−1400 AD) from Point Barrow in Alaska, that is, an Arctic environment well appropriate to the preservation of organic compounds. Considering that seals and whales were the principal Eskimo feeding sources, replicate samples were prepared to optimize the analytical methodology; that is, muscle tissues and blubbers of harbor seal (P. vitulina), gray seal (H. grypus), ringed seal (P. hispida), and beluga whale (D. leucas) species were impregnated and/or heated on modern ceramics to mimic the Eskimo culinary process. The proteomics analytical methodology was based on the following steps: protein extraction from crushed potsherd (25 mg of starting material) was performed with 1% TFA and sonication step, proteins were then denaturated (guanidine), reduced (dithiothreitol), alkylated (iodoacetamide), and hydrolyzed (trypsin). A nanoLC coupled to a highresolution mass spectrometer was used for analysis (for a more 52


Chemical Reviews

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Table 8. Harbor Seal Myoglobin Sequences Identified by nanoLC, nanoESI-Qh-FT-ICR MS/MS from the Protein Extract of the 1200−1400 AD Arctic Potsherd Fragmenta species identified protein myoglobinb

identified sequences SHPETLEKFDKFK YKELGFHG HPAEFGADAQAAMK VETDLAGHGQEVLIR

baikal seal (P. sibirica)

gray seal (H. grypus)

harbor seal (P. vitulina)

X

X X X X

X X X X

X

other marine species c

other species

peptide position

retention time (min)

d e f

36−48 147−154 120−133 18−32

30.3 29.6 30.4 34.3

precursor ions mass/charge

mean error MS/MS/MS

803.417 475.740 722.338 818.936

Proteins in Art, Archaeology, and Paleontology ... - ACS Publications

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