Identification of proteinaceous binders used in artworks by MALDI-TOF mass spectrometry

Proper identification of proteinaceous binders in artworks is essential for specification of the painting technique and thus also for selection of the restoration method; moreover, it might be helpful for the authentication of the artwork. This paper is concerned with the optimisation of analysis of the proteinaceous binders contained in the colour layers of artworks. Within this study, we worked out a method for the preparation and analysis of solid samples from artworks using tryptic cleavage and subsequent analysis of the acquired peptide mixture by matrix-assisted laser desorption/ionisation time of flight mass spectrometry. To make this approach rational and efficient, we created a database of commonly used binders (egg yolk, egg white, casein, milk, curd, whey, gelatine, and various types of animal glues); certain peaks in the mass spectra of these binders, formed by rich protein mixtures, were matched to amino acid sequences of the individual proteins that were found in the Internet database ExPASy; their cleavage was simulated by the program Mass-2.0-alpha4. The method developed was tested on model samples of ground layers prepared by an independent laboratory and then successfully applied to a real sample originating from a painting by Edvard Munch.     

Study of the influencing effect of pigments on the photoageing of terpenoid resins used as pictorial media

Terpenoid resins have been mainly used as components of pictorial varnishes and binding media from ancient times. In such latest instances, the resin is mixed with pigments affecting its physical and chemical properties. A new procedure based on gas chromatography-mass spectrometry (GC/MS) has been applied, in this work, in combination with Fourier transform infrared spectroscopy (FTIR), with the aim of determining the changes undergone by di- and triterpenoid resins employed as components of binding media. The GC/MS method is based on the derivatisation of these resins using trimethylsilylimidazol. Characterization of the main components of the di- and triterpenoid fractions and their oxidised products, occurring in the diterpenoid resin colophony and a triterpenoid Mexical copal (Bursera cuneata (Schl) Engl.), has been achieved. Artificially photoaged specimens of pure resin and binary systems pigment+resin prepared as thin films using lead white, ochre yellow, umber raw and verdigris have also been analysed to study the changes undergone by the di- and triterpenoid components of the resins. Oxidation processes taking place on di- and triterpenoids during photoageing are described. Moreover, the results obtained indicate that pigments have a different effect on the two studied resins. Thus, the presence of pigments induces, in general, a catalysing effect on the oxidative polymerisation reactions in the earliest stages of the natural curing of the paint films whereas a loss of the catalysing effectiveness, in particular, for verdigris containing specimens, is observed during photoageing. Complexation of diterpenoid molecules with copper(II) ions in the earliest stages of the curing has also been evidenced by FTIR analysis.     

Chemical analysis of Gothic mortar from a bridge pier in Roudnice nad Labem (Czech Republic)

In this paper, a Gothic mortar originating from the remains of a bridge pier in Roudnice nad Labem (Czech Republic) was analyzed. The inorganic composition of the mortar showing special mechanical properties was studied using polarizing light microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectrometry, and X-ray diffraction, while the protein content was analyzed by peptide mass mapping using matrix-assisted laser desorption/ionization—time of flight mass spectrometry and nanoliquid chromatography coupled to tandem mass spectrometry. Two types of inorganic binders having a composition similar to mineral wollastonite and hydraulic lime and milk protein additive were detected. We believe that organic and inorganic binders added together to the mortar increase its hardness and firmness in the aquatic environment.     

Collagen-based proteinaceous binder-pigment interaction study under UV ageing conditions by MALDI-TOF-MS and principal component analysis

This study focuses on acquiring information on the degradation process of proteinaceous binders due to ultra violet (UV) radiation and possible interactions owing to the presence of historical mineral pigments. With this aim, three different paint model samples were prepared according to medieval recipes, using rabbit glue as proteinaceus binders. One of these model samples contained only the binder, and the other two were prepared by mixing each of the pigments (cinnabar or azurite) with the binder (glue tempera model samples). The model samples were studied by applying Principal Component Analysis (PCA) to their mass spectra obtained with Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). The complementary use of Fourier Transform Infrared Spectroscopy to study conformational changes of secondary structure of the proteinaceous binder is also proposed. Ageing effects on the model samples after up to 3000?h of UV irradiation were periodically analyzed by the proposed approach. PCA on MS data proved capable of identifying significant changes in the model samples, and the results suggested different aging behavior based on the pigment present. This research represents the first attempt to use this approach (PCA on MALDI-TOF-MS data) in the field of Cultural Heritage and demonstrates the potential benefits in the study of proteinaceous artistic materials for purposes of conservation and restoration.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a tool for fast identification of protein binders in color layers of paintings

Identification of materials in color layers of paintings is necessary for correct decisions concerning restoration procedures as well as proving the authenticity of the painting. The proteins are usually important components of the painting layers. In this paper it has been demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) can be used for fast and reliable identification of proteins in color layers even in old, highly aged matrices. The digestion can be easily performed directly on silica wafers which are routinely used for infrared analysis. The amount of material necessary for such an analysis is extremely small. Peptide mass mapping using digestion with trypsin followed by MALDI-TOFMS and identification of the protein was successfully used for determination of the binder from a painting of the 19th century.     

Innovative technique for the direct determination of proteins in calcified aortic valves

Aortal valve mineralization very frequently causes a genesis of aortic stenosis, which is the most often surgically treated heart disease. Hydroxyapatite deposits have been identified as one of the causes leading to the loss of elasticity of the aortic valves. It is known that phosphates/calcium is accumulated in valve tissues during mineralization, but the mechanism of this process remains unclear. The work is focused mainly on the study of protein composition of mineralized aortic valves by nano-liquid chromatography electrospray ionization in a quadrupole orthogonal acceleration time-of-flight mass spectrometry. New methodological approach based on direct enzymatic digestion of proteins contained in hydroxyapatite deposits was developed for the study of pathological processes connected with osteogenesis. Our objectives were to simplify the traditional analytical protocols of sample preparation and to analyze the organic components of the explanted aortic valves for significant degenerative aortic stenosis. The study of aortic valve mineralization on the molecular level should contribute to understanding this process, which should consequently lead to effective prevention as well as to new ways of treatment of this grave disease.

Analysis of organic colouring and binding components in colour layer of art works

Two methods of analysis of organic components of colour layers of art works have been tested: IR microspectroscopy of indigo, Cu-phthalocyanine, and Prussian blue, and MALDI-TOF-MS of proteinaceous binders and a protein-containing red dye. The IR spectra distortion common for smooth outer surfaces and polished cross sections of colour layer of art works is suppressed by reflectance measurement of microtome slices. The detection limit of the three blue pigments examined is ~0.3 wt% in reference colour layers in linseed oil binder with calcite as extender and lead white as a drying agent. The sensitivity has been sufficient to identify Prussian blue in repaints on a Gothic painting. MALDI-TOF-MS has been used to identify proteinaceous binders in two historical paintings, namely isinglass (fish glue) and rabbit glue. MALDI-TOF-MS has also been proposed for identification of an insect red dye, cochineal carmine, according to its specific protein component. The enzymatic cleavage with trypsin before MALDI-TOF-MS seems to be a very gentle and specific way of dissolution of the colour layers highly polymerised due to very long aging of old, e.g. medieval, samples.     

A Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Method for the Identification of Anthraquinones: the Case of Historical Lakes

This study deals with the identification of anthraquinoid molecular markers in standard dyes, reference lakes, and paint model systems using a micro-invasive and nondestructive technique such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-ToF-MS). Red anthraquinoid lakes, such as madder lake, carmine lake, and Indian lac, have been the most widely used for painting purposes since ancient times. From an analytical point of view, identifying lakes in paint samples is challenging and developing methods that maximize the information achievable minimizing the amount of sample needed is of paramount importance. The employed method was tested on less than 0.5 mg of reference samples and required a minimal sample preparation, entailing a hydrofluoric acid extraction. The method is fast and versatile because of the possibility to re-analyze the same sample (once it has been spotted on the steel plate), testing both positive and negative modes in a few minutes. The MALDI mass spectra collected in the two analysis modes were studied and compared with LDI and simulated mass spectra in order to highlight the peculiar behavior of the anthraquinones in the MALDI process. Both ionization modes were assessed for each species. The effect of the different paint binders on dye identification was also evaluated through the analyses of paint model systems. In the end, the method was successful in detecting madder lake in archeological samples from Greek wall paintings and on an Italian funerary clay vessel, demonstrating its capabilities to identify dyes in small amount of highly degraded samples.

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Proteotypic peptides of hairs for the identification of common European domestic and wild animal species revealed by in-sample protein digestion and mass spectrometry analysis

The aim of this work is to offer an alternative or complementary analytical tool to the time-consuming and expensive methods commonly used for the recognition of animal species according to their hair. The paper introduces a simple and fast way for species differentiation of animal hairs called in-sample digestion. A total of 10 European animal species, including cat, cow, common degu, dog, fallow deer, goat, horse, sika deer, rabbit, roe deer, and 17 different breeds of dogs were examined using specific tryptic cleavage directly in hair followed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-electrospray ionization quadrupole time of flight. Principal component analysis was used for the subsequent mass spectrometric data evaluation. This novel approach demonstrates the ability to distinguish among individual animal species, which is supported by finding characteristic m/z values obtained by the mass spectrometry for each animal species. The approach was successfully tested on two “blind” samples. On the other hand, the attempt to distinguish among hairs of different dog breeds has not been successful due to the very similar protein composition and their amino acid sequences.     

Verification of cheeses authenticity by mass spectrometry

Cheeses are a group of fermented dairy products that are produced all over the world in various forms and flavours. Milk, especially sheep or goat milk, is still regarded as an expensive raw material in the world, which makes milk and milk products highly attractive as a fraud target. Most often, such fraud includes partial or complete substitution with cheaper sorts of milk (e.g. bovine milk). The aim of this work was to verify the authenticity of 27 cheeses commonly emerging on the Czech food market. The cheeses were distinguished on the basis of milk animal species origin. For this purpose, two mass spectrometry techniques were used: matrix‐assisted laser desorption/ionization with time of flight mass spectrometry together with principal component analysis method and liquid chromatography coupled with electrospray ionization and quadrupole time‐of‐flight mass spectrometry. The results were a partial success, because the cheeses could only be partially distinguished with the first mass spectrometry technique probably because of the influence of some protein additive materials in cheeses. The second technique allowed for collecting higher quality results and thus appears to be highly suitable for the research task.