PROTEOMICS AND MASS SPECTROMETRY FOR ARCHAEOLOGY AND PALEONTOLOGY

The preservation of cultural heritage is central for protecting a sense of who we are, a meaningful reference in our culturally diverse world. (1) Efforts to preserve resources on cultural heritage have gained new momentum throughout the world nowadays. The protection of our cultural heritage is a cultural but also economical process. (1) It is nowadays broadly recognized that chemical analysis of materials can contribute to this process with important and often crucial information that are helpful for the study, preservation, or restoration of any kind of object of cultural heritage ranging from works of art to archaeological findings. The identification of materials in and on archaeological objects, monuments or artworks can open a window to the past for archaeologist and historians, and simultaneously helps in paving a safe way to the future, by allowing the conservators to make informed decisions regarding suitable preservation strategies. (2) What has became increasingly important today is: WHAT (identification) has been found and WHERE (provenience). (3) To start with, it is important to record what has been found, before you can delve on the questions about the provenance of the artefacts or the technology of how they were made, (4) and, in this perspective is also important to understand the chemical composition of each remain. (3) Most artefacts are composed of complex mixtures of different molecules having a wide range of chemico-physical properties. Most of them are at a trace level, whilst others are in high amounts. (4) The main challenge in this type of work is the incredible variety and heterogeneity of the materials encountered. (5) Some of them are facing deterioration, corrosion or destruction due to human activity as well as natural causes. Over the centuries, these factors increase and it is becoming more and more important to plan appropriate protection measures to save and preserve timeless evidences of human being (6). If we are able to identify natural substances and their degradation products, we can shed light on the nature and the origin of the material employed and on the state of conservation, as well as on the degradation process itself. Furthermore, analyses need to be as less invasive as possible. Moreover, samples are complex and heterogeneous. Therefore, it is essential to develop analytical protocols, which are minimally invasive and extremely high sensitive. (7) To address these demanding analytical challenges, it is necessary to develop and use powerful and versatile analytical methodologies such as those based on mass spectrometry. These techniques offer unique means for advanced and microdestructive characterization of a wide spectrum of materials in archaeological findings, monuments, and works of art. (4) My PhD project was focused on the development and optimization of chemical and proteomic analytical strategies based on advanced mass spectrometric techniques for archaeological remains. The study of archaeological remains ranges from the molecular characterization of vessels or potteries’ content to the analysis of organic remains such as human and animal tissues, for example bones, but also painting binders or vessels decorations. After a brief introduction, a description of the Archaeological materials herein analysed is reported in Chapter 2, since this is the basis of the scientific interest in the search of new investigation procedures. Since many of the challenges in this field arise from the complex composition and alteration pathways of the materials considered, this Chapter also provides the information of organic materials used, focusing the attention not only on protein-based materials. To understand the potential of proteomics and mass spectrometric techniques applied on Archaeological objects, a description of the common methods is given in Chapter 3. Moreover, to help the understanding of the proteomic strategies developed and the various mass spectrometric methods applied, in this Chapter the main concepts in mass spectrometric instrumentation are also discussed. Chapter 4 describes the development of a unique method that could be considered, efficient and applicable to different kinds of samples that contain proteinaceous materials. In the chapters 5 to 8, a description of different study cases demonstrate the success of the experimental approaches. The project was based on the identification and molecular characterization (by looking at chemical modification) of proteins and other organic molecules in archaeological finds like amphorae, tools and bones after optimization of analytical protocols and mass spectrometry approaches. Specifically, the study was focused on the aging and degradation processes, defining the molecular markers of conservation state in human bones by studying different typologies of burials. Specific attention was paid to human bones from the excavations of Pompeii and Herculaneum. Moreover, the project was focused also to the characterization of embalming materials used in animal mummification process in Sudan. Another case of study deals with the identification of the animal species of a textile membrane thread from a drape found in the tomb of Emperor Henry VII of Luxembourg. As a further section of my project, I worked to the development of a dedicated peptide database and targeted MS method (Multiple Reaction Monitoring, MRM), for the identification of binders in paintings as described in Chapter 9. The need to develop this method comes from the intrinsic analytical disadvantages due to the complexity of these unusual samples, in terms of chemical composition, presence of contaminating protein-based materials, as well as the high levels of molecular damage found in ancient samples. This method can be considered the first example of the application of targeted proteomics to samples in the field of cultural heritage that will allow an increase in sensitivity of protein detection in complex and rather contaminated samples. The improvement in selectivity and sensitivity achieved with the targeted approach in respect to classical profiling experiments could pave the way to the possible application of this most advanced analytical strategy in the field of archaeology and artworks. References 1. EkwelemVO,OkaforVN,UkwomaSC (2011) Preservation of Cultural Heritage: The Strategic Role of the Library and Information Science Professionals in South East Nigeria Library Philosophy And Practice562 2. Zhao W, Cioffi R, Maglioccola F, Catuogno R, Colangelo F, Russo L (2010) New technology and materials on the conservation of cultural heritage. Diagnosis for the conservation and valorization of cultural heritage (1) 319-321. 3. Gunneweg J. (2016) Clay, Technology and Qumran Pottery: What to verify and publish ? Researchgate. 4. Colombini MP, Modugno F (2009) Organic mass spectrometry in art and archaeology. Wiley, New York 5. Jakes KA (2002) Archaeological Chemistry. American Chemical Society. 6. Wadsworth C, Buckley M (2014) Proteome degradation in fossils: Investigating the longevity of protein survival in ancient bone. Rapid Commun. Mass Spectrom (28) 605–615. 7. Vinciguerra R, De Chiaro A, Pucci P, Marino G, Birolo L (2015) Proteomic strategies for cultural heritage: form bones to paintings. Microchem. J. (126) 341–348

Proteomic strategies for cultural heritage: From bones to paintings

In recent years, proteomics procedures have become increasingly popular for the characterization of proteinaceous materials in ancient samples of several cultural heritage objects. The knowledge of the materials used in a work of art is crucial, not only to give an insight in the historical context of objects and artists, but also to analyse degradation processes taking place in aged objects and to develop appropriate conservation and/or restoration treatments. However, protocols routinely applied for typical modern samples still need to be fully adapted to take into account the low amount of proteinaceous material, the heterogeneity and the unusual physical state of the samples, as well as the high levels of damage found in ancient samples. This paper deals with some examples of the adaptation of classical proteomic strategies in the analysis of ancient samples to meet the different aims in the cultural heritage field

Characterization of a surface-active protein extracted from a marine strain of penicillium chrysogenum

Marine microorganisms represent a reservoir of new promising secondary metabolites. Surface-active proteins with good emulsification activity can be isolated from fungal species that inhabit the marine environment and can be promising candidates for different biotechnological applications. In this study a novel surface-active protein, named Sap-Pc, was purified from a marine strain of Penicillium chrysogenum. The effect of salt concentration and temperature on protein production was analyzed, and a purification method was set up. The purified protein, identified as Pc13g06930, was annotated as a hypothetical protein. It was able to form emulsions, which were stable for at least one month, with an emulsification index comparable to that of other known surface-active proteins. The surface tension reduction was analyzed as function of protein concentration and a critical micellar concentration of 2 M was determined. At neutral or alkaline pH, secondary structure changes were monitored over time, concurrently with the appearance of protein precipitation. Formation of amyloid-like fibrils of SAP-Pc was demonstrated by spectroscopic and microscopic analyses. Moreover, the effect of protein concentration, a parameter affecting kinetics of fibril formation, was investigated and an on-pathway involvement of micellar aggregates during the fibril formation process was suggested

Identification of proteinaceous binders in paintings: A targeted proteomic approach for cultural heritage

Abstract Identification of proteins in paintings and polychrome objects is a challenge, which requires the development of tailored analytical approaches. In the present study, a targeted proteomics approach was developed for discriminating among the three most common proteinaceous materials used as paint binders, i.e. milk, egg, and animal glue. In this study a specific database of peptides was created based on tandem MS analyses of tryptic digests of several paint samples collected from a variety of art objects of different ages and conservation conditions. Specific peptide markers of each protein were then selected and monitored by LC-MSMS in Multiple Reaction Monitoring (MRM) ion mode, together with their specific precursor ion-product ion transitions, as defined by their unique amino acid sequence. The developed method enabled a sensitive and reliable detection of the target peptides in a selection of case studies, leading to the unambiguous identification of the proteins used as paint binders. The method showed greatly increased sensitivity compared to currently available strategies

Screening of Fungal Strains for Cellulolytic and Xylanolytic Activities Production and Evaluation of Brewers’ Spent Grain as Substrate for Enzyme Production by Selected Fungi

Brewer’s spent grain (BSG), the solid residue of beer production, is attracting significant attention as raw material for the production of added value substances, since until recently it was mainly used as animal feed or deposited in landfills, causing serious environmental problems. Therefore, this work aimed at developing a bioprocess using BSG as a substrate for the production of cellulases and xylanases for waste saccharification and bioenergy production. Different fungi were analyzed for their cellulolytic and xylanolytic abilities, through a first screening on solid media by assessment of fungal growth and enzyme production on agar containing carboxylmethylcellulose or xylan as the sole carbon source, respectively. The best cellulase and xylanase producers were subjected to quantitative evaluation of enzyme production in liquid cultures. Aspergillus niger LPB-334 was selected for its ability to produce cellulase and xylanase at high levels and it was cultivated on BSG by solid state fermentation. The cellulase production reached a maximum of 118.04 ± 8.4 U/g of dry substrate after 10 days of fermentation, while a maximum xylanase production of 1315.15 ± 37.5 U/g of dry substrate was reached after 4 days. Preliminary characterization of cellulase and xylanase activities and identification of the enzymes responsible were carried out

Identification of proteinaceous binders in paintings: A targeted proteomic approach for cultural heritage

Identification of proteins in paintings and polychrome objects is a challenge, which requires the development of tailored analytical approaches. In the present study, a targeted proteomics approach was developed for discriminating among the three most common proteinaceous materials used as paint binders, i.e. milk, egg, and animal glue. In this study a specific database of peptides was created based on tandem MS analyses of tryptic digests of several paint samples collected from a variety of art objects of different ages and conservation conditions. Specific peptide markers of each protein were then selected and monitored by LC-MSMS in Multiple Reaction Monitoring (MRM) ion mode, together with their specific precursor ion-product ion transitions, as defined by their unique amino acid sequence. The developed method enabled a sensitive and reliable detection of the target peptides in a selection of case studies, leading to the unambiguous identification of the proteins used as paint binders. The method showed greatly increased sensitivity compared to currently available strategies