Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.     

Pyrolysis-gas chromatography/mass spectrometry analysis as a useful tool in forensic examination of automotive paint traces

Pyrolysis (Py)-GC/MS was applied in comparative analysis of polymer binder of 150 automobile clearcoat samples. It was found that binders of identical type and similar infrared spectra can be, in most cases, effectively differentiated, sometimes based only on the presence of peaks of very low intensity originating from minor paint components. The qualitative procedure of pyrograms comparison was developed. The results obtained, enable one to draw the conclusion that Py-GC/MS appears as valuable, very informative analytical technique of car paint samples examination. Providing identification of paint pyrolysis products, it enables discrimination of samples on the basis of their composition. However, in some cases, paint samples were still indistinguishable on the basis of their pyrograms.     

Physico-chemical characterization of protein-pigment interactions in tempera paint reconstructions: casein/cinnabar and albumin/cinnabar

In this work, we characterized paint reconstructions using ovalbumin and casein as binders, and cinnabar (HgS) as a pigment, before and after artificial ageing. Egg and casein are common paint binders that were used historically in the technique of tempera painting. Despite extensive research on the identification of proteinaceous binders in paintings, there is a substantial lack of knowledge regarding the ageing pathway of their protein content, and their chemical interaction with inorganic pigments. Thermogravimetric analysis, infrared spectroscopy and size-exclusion chromatography (SEC) were used to reveal the physico-chemical processes involved in the ageing of proteins in paintings. Taken together, the three techniques highlighted that proteins are subject to both cross-linking and hydrolysis upon ageing, and to a lesser extent, to oxidation of the side chains. Mercury–protein interactions were also revealed using a cold vapour generation atomic fluorescence spectrometer mercury-specific detector coupled to SEC. The study clearly showed that HgS forms stable complexes with proteins and acts as a sensitizer in cross-linking, hydrolysis and oxidation.     

Surface-enhanced Raman spectroscopy analysis of house paint and wallpaper samples from an 18th century historic property

Conservation efforts for heritage buildings require a substantial knowledge of the chemical makeup of materials that were used throughout the lifetime of the property. In particular, conservators are often concerned with the identification of colorants used in both interior and exterior wall treatments (paint, wallpaper, etc.) in order to gain perspective into how the building may have appeared during a certain time period in its existence. Ideally, such an analysis requires a technique that provides molecular level information as to the identity of the colorant as well as other sample components (binders, fillers, etc.), which is useful for dating purposes. In addition, the technique should be easily applied to paint layer samples which can be extremely thin and fragile. Herein we report the surface-enhanced Raman spectroscopy (SERS) analysis of paint and wallpaper samples taken from exterior and interior surfaces of a historic building. Several pigments were identified in the samples, which ranged from early inorganic pigments (lead white, barium sulfate, calcium carbonate, anhydrous chromium(III) oxide) which have been used in house paints for centuries, to a more modern pigment (phthalocyanine blue), developed in the middle of the 20th century. This analysis highlights the usefulness of SERS in such a conservation effort, and demonstrates for the first time pigment identification in house paints and wallpaper using SERS, which has far-reaching implications not only in the field of conservation, but also in forensics, industrial process control, and environmental health and safety.     

GC-MS characterization of proteinaceous and lipid binders in UV aged polychrome artifacts

This paper presents the most significant results obtained from the characterization of protein and lipid binders in a broad range of reference paint materials prepared and stored at Opificio delle Pietre Dure in Florence (Italian Ministry of the Cultural Heritage, Italy). The amino acid distribution for protein binders and the fatty acid distribution for lipid binders were determined by gas chromatography-mass spectrometry before and after being artificially aged through exposure to UV light at 254 nm or 366 nm for different time periods in a climatic chamber (20°C and 80% RH). The results were compared with those relevant to old paintings of different ages, and showed that UV light aging does not significantly affect the amino acid profile of protein binders. Consequently, protein binders in old paintings can be reliably identified by comparing the amino acid composition with that of reference paint materials which have not been aged. However, the composition of lipid binders is substantially affected by UV irradiation, leading to a lowering of oleic acid and the formation of azelaic acid and other dicarboxylic acids including oxalic acid. An oleic to stearic acid ratio of less than 0.5 was observed in all the samples of works of art, and this parameter can be used to evaluate the extent of the artificial aging process. The formation of oxalic acid was also observed starting with pure unsaturated fatty acids, thus supporting the chemical origin of oxalate patina.     

Multivariate chemical mapping of pigments and binders in easel painting cross-sections by micro IR reflection spectroscopy

Paintings are composed of superimposed layers of inorganic and organic materials (pigments and binders). Knowledge of the stratigraphic sequence of these heterogeneous layers is fundamental for understanding the artist’s painting technique and for conservation issues. In this study, micro-IR mapping experiments in reflection mode have been carried out on cross-sections taken from simulations of ancient easel paintings. The objective was to locate both organic binders and inorganic pigments. Chemical maps have been re-constructed using the common approach based on the integration of specific infrared bands. However, owing to the complexity of painting materials, this approach is not always applicable when dealing with broad and superimposed spectral features and with reststrahlen or derivative-like bands resulting from acquisition in reflection mode. To overcome these limitations, principal-component analysis has been successfully used for the re-construction of the image, extracting the relevant information from the complex full spectral data sets and obtaining reliable chemical distributions of the stratigraphy materials. Different pigment–binder combinations have been evaluated in order to understand limitations and strengths of the approach. Finally, the method has been applied for stratigraphic characterization of a cross-section from a 17th century wooden sculpture identifying both the original paint layer and the several overpaintings constituting the complex stratigraphy.     

Identification of the chemical inventory of different paint types applied in nuclear facilities

The floors, concrete walls and many of the metal surfaces in nuclear power plant containments are coated with zinc primers or paint films to preserve the metal surfaces and simplify decontamination in the containment after the occurrence of a severe nuclear incident or accident. A chemical examination of paint films from different nuclear installations out of operation, as well as current operating ones, reveals that different types of paints are used whose composition can vary significantly. Results obtained for one type of paint at a certain nuclear site are in most cases unlikely to be comparable with sites painted with another type of paint. During normal operation and particularly during nuclear accidents, the paints will degrade under the high temperature, steam and irradiation influence. As paint and its degradation products can act as sources and depots for volatile iodine compounds, the type and aging conditions of the paint films will have a significant impact on the source term of the volatile fission product iodine. Thus, great care should be taken when extrapolating any results obtained for the interaction of radioactive iodine with one paint product to a different paint product. The main focus of the study is a comparison of the chemical profile of paint films applied in Swedish nuclear power plants. Teknopox Aqua V A, an epoxy paint recently used at Ringhals 2, and an emulsion paint used in the scrubber buildings of Ringhals 1–4 are compared with a paint film from Barsebäck nuclear power plant unit 1 that had been aged under real reactor conditions for 20 years. In addition, two paint films, an emulsion and a gloss paint, used in an international nuclear fuel reprocessing facility, are compared with the paints from the Swedish nuclear power plants.     

A simple protocol for Matrix Assisted Laser Desorption Ionization- time of flight-mass spectrometry (MALDI-TOF-MS) analysis of lipids and proteins in single microsamples of paintings

A simple protocol, based on Bligh–Dyer (BD) extraction followed by MALDI-TOF-MS analysis, for fast identification of paint binders in single microsamples is proposed. For the first time it is demonstrated that the BD method is effective for the simultaneous extraction of lipids and proteins from complex, and atypical matrices, such as pigmented paint layers. The protocol makes use of an alternative denaturing anionic detergent (RapiGest™) in order to improve efficiency of protein digestion and purification step. Detection of various lipid classes, such as triacylglycerols (TAGs) and phospholipids (PLs), and their oxidation by-products was accomplished, whereas proteins could be identified by peptide mass fingerprinting. The effect of pigments on ageing of lipids and proteins was also investigated.     

The development of a gas chromatographic–mass spectrometric analytical procedure for the determination of lipids,proteins and resins in the same paint micro-sample avoiding interferences from inorganic media

This paper presents a GC–MS analytical procedure for determining proteinaceous materials, glycerolipids, natural waxes and terpenoid resins in the same paint micro-sample. The procedure is also reliable when high amounts of interfering inorganic pigments, dryers and charges are present. The characterisation of proteinaceous binders in a paint sample can be subject to analytical interferences by inorganic materials. Such materials may form complexes with functional groups of proteins, thus preventing their efficient derivatisation, which is necessary prior to GC analysis. For this reason an analytical procedure has been developed based on two extractions and a clean-up step, in order to obtain two fractions: a lipid-resinous fraction and a proteinaceous fraction. The lipid-resinous fraction is subjected to salification/saponification assisted by microwaves, followed by acidification, extraction, derivatisation and GC–MS analysis. The proteinaceous fraction is analysed by GC–MS after hydrolysis and derivatisation of the freed amino acids. The desalting step is applied before the hydrolysis, and is based on the use of the monolithic sorbent tip technology with a C4 stationary phase. Reference paint replicas of egg, casein and animal glue were prepared with and without several metals containing pigments, and used to develop and validate the analytical procedure. The procedure proved to be efficient in desalting the proteinaceous materials both from cations and anions. Although non quantitative, it is reliable in the analysis of samples whose content of extractable proteins is <1 μg, thus showing it to be suitable for the characterisation of paint samples. An example of how the analytical procedure was used to characterise a sample from a 15th century panel painting is also discussed.     

Identification of protein binders in works of art by high-performance liquid chromatography–diode array detector analysis of their tryptic digests

Proteins in works of art are generally determined by the relative amounts of amino acids. This method, however, implies a loss of information on the protein structure and its modifications. Consequently, we propose a method based on the analysis of trypsin digests using high-performance liquid chromatography (HPLC) UV diode array detection (DAD) for painting binder studies. All reaction steps are done in the same vial; no extraction methods or sample transfer is needed, reducing the risk of sample losses. A collection of pure binders (collagen, ovalbumin, yolk and casein) as well as homemade and historical paint samples have been investigated with this method. Chromatograms of unknowns at 214 nm and 280 nm are compared with those of the reference samples as a fingerprint. There is a good agreement between many peptides, but others seem to have been lost or their retention time shifted due to small compositional changes because of ageing and degradation of the paint. The results are comparable with the results of other techniques used for binder identification on the same samples, with the additional advantage of differentiation between egg yolk and glair.     

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. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of paint cross-sections: a combined multivariate approach for the interpretation of μATR-FTIR hyperspectral data arrays

The present research is aimed at introducing a suitable approach for the exploitation of the hyperspectral data obtained by μATR-FTIR analyses of paint cross-sections. The application of principal component analysis for chemical mapping is well-established, even if a very limited number of applications to μFTIR data have been reported so far in the field of analytical chemistry for cultural heritage. Moreover, in many cases, chemometric tools are under-utilized and the outcomes under-interpreted. As a consequence, results and conclusions may be considerably compromised. In an attempt to overcome such drawbacks, the present work is proposing a comprehensive and efficient procedure based on an interactive brushing approach, which combines the structural information of the score scatter plots and the spatial information of the principal component (PC) score maps. In particular, the study demonstrates not only how the multivariate approach may provide more information than the univariate one, but also how the integration of different chemometric tools may allow a more comprehensive interpretation of the results with respect to the studies up to now reported in the literature. The examination of the average spectral profile of each score cluster, jointly with the loading analysis, is functional to characterize each area investigated on the basis of its spectral features. A multivariate comparison with spectra of standard compounds, projected in the PC score space, helps in supporting the chemical identification. The approach was validated on two real case studies.     

Single and multiplexed immunoassays for the chemiluminescent imaging detection of animal glues in historical paint cross-sections

The characterization of the organic components in a complex, multilayered paint structure is fundamental for studying painting techniques and for authentication and restoration purposes. Proteinaceous materials, such as animal glue, are of particular importance since they are widely used as binders, adhesives and for gilding. Even though proteins are usually detected by chromatographic and proteomic techniques, immunological methods represent an alternative powerful approach to protein analysis thanks to the high specificity of antigen–antibody reactions. Our previous studies demonstrated that ovalbumin and casein could be localized in paint cross-sections with high sensitivity and good spatial resolution (i.e. within the single painting layers) by using chemiluminescent (CL) immunochemical microscope imaging. In the present research work, we describe for the first time the immunolocalization of collagen (the main protein of animal glue) in paint cross-sections by CL imaging microscopy. Two different analytical protocols have been developed, allowing either the detection of collagen or the simultaneous detection of collagen and ovalbumin in the same paint sample. The assays were used to detect collagen and ovalbumin in cross-sections from model samples and historical paintings (a wall painting dated to 1773–1774 and a painted wood panel of the Renaissance period) in order to achieve information on paint techniques and past restoration interventions.

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).     

How to control dirt pick-up of exterior coatings

The composition of dirt and the tendency of coatings to pick up dirt are determined largely by the location. The factors affecting the tendency of coatings to pick up dirt are varied and complex. That is why the problem of dirt pick-up on exterior coatings can only be resolved satisfactorily by optimisation of the paint formulation as a whole (PVC, type of formulation, surface texture) and of all the raw materials (binders, pigments, fillers, additives).     

RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process. We present here a method, RAMPED-UP NMR, (Rapid Analysis and Multiplexing of Experimentally Discriminated Uniquely Labeled Proteins using NMR) which generates simple spectra which are easy to interpret and allows several proteins to be screened simultaneously. In this method, the proteins to be screened are uniquely labeled with one amino acid type. There are several benefits derived from this unique labeling strategy: the spectra are greatly simplified, resonances that are most likely to be affected by binding are the only ones observed, and peaks that yield little or no information upon binding are eliminated, allowing the analysis of multiple proteins easily and simultaneously. We demonstrate the ability of three different proteins to be analyzed simultaneously for binding to two different ligands. This method will have significant impact in the use of NMR spectroscopy for both the lead generation and lead optimization phases of drug discovery by its ability to increase screening throughput and the ability to examine selectivity. To the best of our knowledge, this is the first time in any format that multiple proteins can be screened in one tube.     

Ideal für kleine Künstler: Fingerfarben

Finger paint is a remarkable product of modern chemistry. The main components are polysaccharides of plant or semi‐synthetic origin or non‐toxic polyglycols as binders, mineral carbonates, or oxides as fillers, pigments, and/or dyes, and preservatives that delay microbiological spoilage. Since 2002 the composition has been strictly controlled under European Law especially in regard to which pigments, dyes, and preservatives are allowed. All constituents in finger paint can be regarded as safe and most of them are used in cosmetics and as food additives. In summary, finger paint is totally safe in the hands of our children.     

The biodeterioration of synthetic resins used in conservation

Synthetic resins have been extensively employed by artists in their works of art, e.g. as paint binders, or by conservators for conservation treatments, e.g. as stone consolidants and protectives. It is generally thought that synthetic resins are less prone to chemical, physical and biological deterioration than other organic products but there are many articles in the scientific literature and some reports in the conservation of cultural heritage literature claiming that microorganisms are capable of degrading synthetic resins. This paper reviews the researches on the biodeterioration of synthetic resins used in the conservation of cultural heritage, including stone, painting and textile materials, carried out in the last fifty years.     

Proteomic strategies for the identification of proteinaceous binders in paintings

The identification of proteinaceous components in paintings remains a challenging task for several reasons. In addition to the minute amount of sample available, complex and variable chemical composition of the paints themselves, possible simultaneous presence of several binders and contaminants, and degradation of the original materials due to aging and pollution are complicating factors. We proposed proteomic strategies for the identification of proteins in binders of paintings that can be adapted to overcome the requirements and difficulties presented by specific samples. In particular, we worked on (1) the development of a minimally invasive method based on the direct tryptic cleavage of the sample without protein extraction; (2) the use of microwave to enhance the enzymatic digestion yield, followed by the analysis of the peptide mixtures by nanoLC-MS/MS with electrospray ionization (ESI). Moreover, as an additional tool to tackle the problem of contaminating proteins, we exploited the possibility of generating an exclusion list of the mass signals that in a first run had been fragmented and that the mass spectrometer had to ignore for fragmentation in a subsequent run. The methods, tested on model samples, allowed the identification of milk proteins in a sample from paintings attributed to Cimabue and Giotto, thirteenth-century Italian masters, decorating the vaults of the upper church in the Basilica of St. Francis in Assisi, Italy.     

Comparative analysis of automotive paints by laser induced breakdown spectroscopy and nonparametric permutation tests

Laser-induced breakdown spectroscopy (LIBS) has been investigated for the discrimination of automobile paint samples. Paint samples from automobiles of different makes, models, and years were collected and separated into sets based on the color, presence or absence of effect pigments and the number of paint layers. Twelve LIBS spectra were obtained for each paint sample, each an average of a five single shot “drill down” spectra from consecutive laser ablations in the same spot on the sample. Analyses by a nonparametric permutation test and a parametric Wald test were performed to determine the extent of discrimination within each set of paint samples. The discrimination power and Type I error were assessed for each data analysis method. Conversion of the spectral intensity to a log-scale (base 10) resulted in a higher overall discrimination power while observing the same significance level. Working on the log-scale, the nonparametric permutation tests gave an overall 89.83% discrimination power with a size of Type I error being 4.44% at the nominal significance level of 5%. White paint samples, as a group, were the most difficult to differentiate with the power being only 86.56% followed by 95.83% for black paint samples. Parametric analysis of the data set produced lower discrimination (85.17%) with 3.33% Type I errors, which is not recommended for both theoretical and practical considerations. The nonparametric testing method is applicable across many analytical comparisons, with the specific application described here being the pairwise comparison of automotive paint samples.