Collagen Fingerprinting of Archaeological Bone and Teeth Remains from Domuztepe, South Eastern Turkey
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Archaeol Anthropol Sci (2011) 3:271–280 DOI 10.1007/s12520-011-0066-z ORIGINAL PAPER Collagen fingerprinting of archaeological bone and teeth remains from Domuztepe, South Eastern Turkey Mike Buckley & Sarah Whitcher Kansa Received: 7 December 2010 /Accepted: 21 March 2011 /Published online: 7 May 2011 # Springer-Verlag 2011 Abstract We applied a recently developed method of collagen fingerprinting. We also show that the collagen collagen peptide mass fingerprinting for taxonomic identi- extracted from dentine is equally amenable to the proposed fication in faunal remains to 111 specimens from the methodology as bone collagen. Neolithic site of Domuztepe in southeastern Turkey. Advances on the published technique allow us to move Keywords Collagen fingerprinting . MALDI . beyond the key domesticated fauna (sheep, goat, pig and Archaeological proteomics . ZooMS cattle) to include non-domesticates (deer and gazelle, as well as humans). As the animal economy of Domuztepe was dominated by domestic sheep and goats, a large Introduction number of the samples tested were morphologically indistinguishable sheep/goat remains. A number of samples The domestication and exploitation of animals represents represented elements that provide information on age (i.e., one of the critical stages in the changing human articulation mandibles) or sex (pelvis); thus, this represents the first with the living world (Barker et al. 1988). Sheep and goat major study to attempt to improve kill-off profiles using were two of the first animals domesticated in the Near East and, by the 8th millennium BC, became the primary animals exploited by Neolithic people in this region (Peters et al. 2005). However, as sheep and goat bones are Electronic supplementary material The online version of this article notoriously difficult to distinguish, particularly with young (doi:10.1007/s12520-011-0066-z) contains supplementary material, individuals or fragmented assemblages, zooarchaeologists which is available to authorized users. frequently aggregate them into a single ‘sheep/goat’ M. Buckley (*) category. While aggregation increases sample size and Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, provides a general impression of the age and sex compo- 131 Princess Street, sition of the combined sheep/goat population, it is much Manchester M1 7DN, UK e-mail: [email protected] more desirable to separate the sheep from the goats in order to reveal differential exploitation patterns that can be M. Buckley reflected in the harvest profiles of sheep and goats BioArCh, Department of Biology, Archaeology and Chemistry, separately. Though their bones are difficult to distinguish, University of York, Heslington, domestic sheep (Ovis aries) and goats (Capra hircus) are York YO10 5YW, UK dissimilar in many ways: they have different feeding patterns and requirements with regards to husbandry S. W. Kansa practices, they tolerate different environmental conditions The Alexandria Archive Institute, 125 El Verano Way, and they provide a variety of products. The ability to San Francisco, CA 94127, USA distinguish the archaeological bones of sheep and goats, 272 Archaeol Anthropol Sci (2011) 3:271–280 thus, has profound implications for understanding prehis- peptides are fractionated using solid-phase extraction (SPE) toric economies and the differential utilisation of these C18 pipette tips to a lesser extent than the method used species by humans. previously (Buckley et al. 2010), resulting in a greater Zooarchaeologists working with material from the Near number of peptides being utilised as markers. A previously East tend to rely on a few key studies that present identified set of collagen peptide markers for a wide range of morphological distinctions between sheep and goat bones mammalian species (Buckley et al. 2009) to which peptide (e.g. Boessneck 1969; Payne 1969; Payne 1985; Prummel markers for gazelle (Gazella gazella) have been added, are and Frisch 1986), although more recent studies have both used to identify challenging samples (i.e. highly fragmented expanded and tested these earlier works (Halstead et al. specimens or those with few distinguishing morphological 2002; Helmer 2000; Zeder and Lapham 2010; Zeder and criteria present) including bones and teeth from Domuztepe, a Pilaar 2010). The methodologies described in many of large settlement in south east Turkey, where excavations to these early studies have become commonplace in Near date have produced material culture dating from ca. 5,800 to Eastern faunal analysis. Nevertheless, the reliability of an ca. 5,450 cal. BC; (Campbell et al. 1999). individual researcher’s determination will largely be depen- The animal economy of Domuztepe was dominated by dent on the preservation of the specimen, the age of the domestic sheep and goats, whose bones make up more than animal at death (where very young animals can lack clear 50% of the entire analysed assemblage to date (Kansa In morphological markers) and, finally, the analyst’s skill and prep.). Management of sheep and goat herds varies depend- confidence in making the determination. Recent studies ing on the needs of the communities keeping the animals. have demonstrated that certain morphological criteria on While both sheep and goat certainly provided a reliable long bones are, indeed, more reliable than others and can be source of meat, sinews, and hides to the people of used to confidently distinguish between sheep and goat, Domuztepe, they also may have been used to provide milk, particularly for complete, adult specimens where multiple hair, wool and dung. These ‘secondary products’, which criteria are present (Buckley et al. 2010; Zeder and Lapham can be taken from the animal while it is still alive (Sherratt 2010). In the absence of clear morphological markers on 1983), differ between taxa, and herds were managed in fragmented specimens, however, there is a common desire for specific ways to target certain products. However, the a cheap and reliable molecular method that still succeeds in difficulty of distinguishing sheep and goats often obscures thermally degraded samples such as those found in the Near some of the finer-grained differences in the harvesting East, and one such method is presented here. profiles of these two taxa. Other methods of differentiation have focussed on dietary In our previous report (Buckley et al. 2010), we signatures such as stable carbon isotopes (Balasse and demonstrated the ability to distinguish between sheep and Ambrose 2005) and tooth wear (Grant 1982; Grine et al. goat using a single collagen peptide marker. This report 1987), but due to influences in dietary response cannot be seeks to show the potential for an alternative method, using considered objective approaches. Ancient DNA analysis does a more informative collagen fingerprint that not only offer a clearly objective approach to species identification but distinguishes between sheep and goats, but also between rarely succeeds in Near Eastern samples of such antiquity many other taxa including humans. A human signature has (Bar-Gal et al. 2003; Loreille et al. 1997). Proteins on the been included because in some circumstances, such as the other hand, have long been known as the dominant ‘Death Pit’ at Domuztepe, where >10,000 remains from biomolecules in modern and ancient bone (Ambler and humans and domestic food animals were highly fragmented Daniel 1991), where collagen (type 1) is by far the most and frequently mixed, distinction between human and non- abundant, taking up approximately 90% by weight of the human vertebrate remains is extremely difficult. However, proteins in bone and known to persist with extraordinary the focus of this paper, the collagen fingerprinting method- longevity under these conditions. We have previously ology, is applied to a much larger number of morpholog- reported a method (ZooMS; Buckley et al. 2010) whereby a ically unidentifiable sheep/goat specimens, some with single collagen peptide that is capable of distinguishing estimated age and known sex, in order to derive support between sheep and goat collagen can be reproducibly isolated for the proposed methods of animal husbandry for the site. via their interactions with standard sample preparation C18 columns, and analysed by matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) mass spectrometry. Experimental Here, we report the application of a simple alternative that produces peptide markers diagnostic of a range of taxa Materials (Buckley et al. 2009), not only key domesticate fauna such as sheep, goat, pig and cattle, but also non-domesticates Hydrochloric acid (HCl), ammonium bicarbonate, and including deer and gazelle, as well as humans. In this variant, acetonitrile (ACN) were obtained from BDH (UK). Archaeol Anthropol Sci (2011) 3:271–280 273 Sequencing grade trypsin, trifluoroacetic acid (TFA), mass TFA, followed by one bed volume of 0.1% TFA. Following spectrometric standards and α-cyano-4-hydroxycinnamic enzymatic digestion, the samples were centrifuged at acid were purchased from Sigma-Aldrich (UK). C18 SPE 13,000×g for 10 min and the supernatant acidified to 0.1% pipette tips were purchased from OMIX (UK). The six TFA and applied to the pipette tip. Following two wash steps MALDI-ToF-MS calibration peptides were purchased from (100 μL 0.1% TFA), a stepped gradient of increasing ACN Applied Biosystems. concentration (100 μL fractions of 10% and 50% ACN in 111 archaeological samples from Neolithic Domuztepe 0.1% aqueous TFA)