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Identification of protein residues in several materials found at Blombos Cave

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CaseHeader

Status: 

HeritageAuthority(s): 

Case Type: 

ProposalDescription: 

Overview The processes that have led human populations of the past to develop the cultural innovations that make us different from our phylogenetically closer relatives (e.g., making composite tools, creating symbolic items, developing numerical symbol systems etc.) are the subject of intense debate. The emergence of cultural innovations implying the use of organic material (resins for hafting, poison for hunting, binders to produce paints, etc.) are highly relevant to these debates since the preparation of such compounds is often cognitively demanding and complex to transmit to new generations. We know that complex organic compounds were produced by both Middle Stone Age (MSA) populations in Africa and Neanderthals in Europe and the Near East since at least 180 ka, but evidence for these innovations remains circumstantial. One reason for this is that finding and identifying organic compounds are particularly challenging. Ancient protein residues are degraded and mostly present in small quantities and incorporated in a complex matrix of organic and inorganic materials such as pigments, degradation products and contaminants. Analysing these types of materials requires dedicated and sensitive analytical techniques. Palaeoproteomics provides information about the taxonomy and the tissue of the organism from which the protein(s) are derived. No other technique is able to provide such information. The Palaeoproteomic research unit at the University of Copenhagen, Denmark, have already demonstrated that it is possible to extract and analyse ancient proteins from a wide variety of tissues [1–3] and dated to a million years [4]. In this proposal we aim to further our understanding of organic compounds possibly present in the ochre mixtures associated with materials discovered at the Blombos Cave site [5]. Objective of your research To identify protein residues from ochre mixtures associated with several materials discovered at Blombos Cave archaeological site. NOTE. If the above aim cannot be met because protein residues are unidentifiable then alternative aims will be formulated, looking at other potentially suitable archaeological materials from Blombos Cave or Klipdrift Shelter or Klipdrift Cave. Methodology Since the materials to be studied are up to ~100,000 years old, low preservation of organic residues is expected. Thus, the most advanced palaeoproteomics methods for the recovery and analysis of ancient protein residues will be used, following the robust experimental protocols developed and routinely used at the University of Copenhagen. To analyse the artefacts using palaeoproteomic techniques, a small quantity of material (see details below) is taken from the sample mechanically. We will sample areas of the artefacts which are most likely to preserve organic residues, such as those not exposed to the external contaminants. When possible, 2-3 locations on each artefact will be sampled. Proteins will be extracted from sub-samples of the samples removed from the artifacts and then digested into smaller fragments, i.e. peptides, using several chemical solutions/solvents (Please find the list of reagents in [1]). The peptides will be analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS). The amount of mass spectrometric data generated in this way is so large that it needs to be processed using bioinformatic tools such as MaxQuant [6]. Finally, the identification of peptides allows the identification of the proteins present within the sample [7], leading to a taxonomic or tissue attribution. Sample types, sampling locations and minimum sample requirement BBC c. 100 ka MSA Toolkit: Orange ring residues from inner surface of toolkit 1 (TK1)— Two spots of the orange residues inside toolkit 1 will be scraped to obtain approximately 100 mg of sample. BBC c. 85-90 ka: Ochre-red sand mixture—About 100 mg red ochre mixture were sampled at the site during excavation. BBC c. 85-90 ka: Shell fragments covered with ochre mixture (O10, O19, and O29)—The whole fragment was collected at the site. The inner surface of the shell will be scraped in order to remove the red ochre mixture for further analysis. BBC c. 85-90 ka: Lithic/ stone piece covered with ochre mixture (L38, L91, and O19 stone piece)—In order to remove the red ochre mixture for further analysis, either the surface of the object will be scraped, or the object will be immersed in a solvent. Soil—Soil surrounding the sampled artefacts will be analysed in parallel with the object itself, using the same laboratory procedure. The rationale is to detect possible contaminants in the soil which, in the absence of soil analysis, could hamper interpretation of data from the samples.

Expanded_Motivation: 

Statement why this study cannot be done in South Africa. Analysing ancient materials is challenging as the amount of organic material preserved is usually very limited. Therefore, these analyses require state-of-the-art palaeoproteomics expertise and laboratory facilities. Palaeoproteomics is a growing field of study and South Africa does not currently have the facilities to carry out the analyses envisaged in this application. Studies show that palaeoproteomics is a very promising tool to detect and characterize protein residues from ancient objects which cannot be carried out through other analytical approaches [2,10].

ApplicationDate: 

Tuesday, July 5, 2022 - 16:37

CaseID: 

18991

OtherReferences: 

ReferenceList: 

CitationReferenceTypeDate Retrieved
M. Mackie, P. Rüther, D. Samodova, F. Di Gianvincenzo, C. Granzotto, D. Lyon, D.A. Peggie, H. Howard, L. Harrison, L.J. Jensen, Palaeoproteomic profiling of conservation layers on a 14th century Italian wall painting, Angew. Chemie Int. Ed. 57 (2018) 7369–7374.
Saturday, June 20, 2020
E. Cappellini, L.J. Jensen, D. Szklarczyk, A. Ginolhac, R.A.R. da Fonseca, T.W. Stafford Jr, S.R. Holen, M.J. Collins, L. Orlando, E. Willerslev, Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins, J. Proteome Res. 11 (2012) 917–926.
Wednesday, July 20, 2022
E. Cappellini, F. Welker, L. Pandolfi, J. Ramos-Madrigal, D. Samodova, P.L. Rüther, A.K. Fotakis, D. Lyon, J.V. Moreno-Mayar, M. Bukhsianidze, Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny, Nature. 574 (2019) 103–107.
Monday, June 20, 2022
B. Demarchi, S. Hall, T. Roncal-Herrero, C.L. Freeman, J. Woolley, M.K. Crisp, J. Wilson, A. Fotakis, R. Fischer, B.M. Kessler, Protein sequences bound to mineral surfaces persist into deep time, Elife. 5 (2016) e17092.
Monday, June 20, 2022
C.S. Henshilwood, F. d’Errico, K.L. van Niekerk, Y. Coquinot, Z. Jacobs, S.-E. Lauritzen, M. Menu, R. García-Moreno, A 100,000-Year-Old Ochre-Processing Workshop at Blombos Cave, South Africa, Science (80-. ). 334 (2011) 219 LP – 222. doi:10.1126/science.1211535.
Monday, June 20, 2022
S. Tyanova, T. Temu, J. Cox, The MaxQuant computational platform for mass spectrometry-based shotgun proteomics, Nat. Protoc. 11 (2016) 2301–2319.
Monday, June 20, 2022
E. Cappellini, A. Prohaska, F. Racimo, F. Welker, M.W. Pedersen, M.E. Allentoft, P. de Barros Damgaard, P. Gutenbrunner, J. Dunne, S. Hammann, Ancient biomolecules and evolutionary inference, Annu. Rev. Biochem. 87 (2018) 1029–1060.
Monday, June 20, 2022
 
 

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