Nontargeted Pattern Recognition in the Search for Pyrolysis Gas Chromatography/Mass Spectrometry Resin Markers in Historic Lacquered Objects.

A differential expression analysis technology developed for linear modeling of gene expression data was used in combination with thermally assisted hydrolysis and methylation gas chromatography/mass spectrometry (THM-GC/MS) to support the analysis of lacquers and varnishes on historical objects. Exudates from tropical trees, such as Manila copal, sandarac, South American copal, and Congo copal, which were frequently used in finishing layers on decorative objects up to the early 20th century, were compared through this approach. Highly discriminating features indicate biomarkers that can help to identify copals in resinous lacquers. The approach allows new, more systematic ways for finding biomarkers in the analysis of lacquered objects of art and varnishes.

Microplastic contamination in gudgeons (Gobio gobio) from Flemish rivers (Belgium).

Plastic pollution is continuously growing on a global scale and emerging as a major environmental hazard. Smaller-sized plastics, so-called microplastics (<5 mm), are considered as being omnipresent throughout the aquatic environment, yet freshwater ecosystems have received little attention so far and are still largely unstudied. Present study aims to expand the current knowledge on microplastics in freshwater systems by documenting the occurrence in the digestive system of fish from 15 rivers at 17 locations in Flanders, Belgium. To increase inter-study comparability and identification accuracy, a more standardized protocol was combined with a conservative approach towards acceptance of microplastic particles. Four rivers were found to have fish containing microplastics. However, no significant differences could be established between the sampling sites. In total 78 specimens of gudgeon (Gobio gobio) have been investigated, 9% of which had ingested at least one microplastic item, thus showing that contamination appears to be limited. Microscopic and spectroscopic analysis showed the microplastics to be from various sources with a diverse range of physical characteristics. Out of the eight items identified as microplastics, seven different polymer types were identified. Although further detailed research is necessary, this preliminary study shows that gudgeons from several Flemish rivers are contaminated with microplastics.

Improved radiocarbon analyses of modern human hair to determine the year-of-death by cross-flow nanofiltered amino acids: common contaminants, implications for isotopic analysis, and recommendations.

RATIONALE: In forensic investigation, radiocarbon ((14)C) measurements of human tissues (i.e., nails and hair) can help determine the year-of-death. However, the frequent use of cosmetics can bias hair (14)C results as well as stable isotope values. Evidence shows that hair exogenous impurities percolate beyond the cuticle layer, and therefore conventional pretreatments are ineffective in removing them. METHODS: We conducted isotopic analysis ((14)C, δ(13)C, δ(15)N and C/N) of conventionally treated and cross-flow nanofiltered amino acid (CFNAA)-treated samples (scalp- and body-hair) from a single female subject using fingernails as a reference. The subject studied frequently applies a permanent dark-brown dye kit to her scalp-hair and uses other care products for daily cleansing. We also performed pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) analyses of CFNAA-treated scalp-hair to identify contaminant remnants that could possibly interfere with isotopic analyses. RESULTS: The conventionally treated scalp- and body-hair showed (14)C offsets of ~21‰ and ~9‰, respectively. These offsets confirm the contamination by petrochemicals in modern human hair. A single CFNAA extraction reduced those offsets by ~34%. No significant improvement was observed when sequential extractions were performed, as it appears that the procedure introduced some foreign contaminants. A chromatogram of the CFNAA scalp-hair pyrolysis products showed the presence of petroleum and plant/animal compound residues, which can bias isotopic analyses. CONCLUSIONS: We have demonstrated that CFNAA extractions can partially remove cosmetic contaminants embedded in human hair. We conclude that fingernails are still the best source of keratin protein for year-of-death determinations and isotopic analysis, with body-hair and/or scalp-hair coupled with CFNAA extraction a close second.

Development of a dedicated peptide tandem mass spectral library for conservation science.

In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art.

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.

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).

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.