Recovering bone and tooth proteins in arid environments using palaeoproteomics -A case study from the Late Stone Age site of Toteng, Botswana

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Recovering bone and tooth proteins in arid

environments using palaeoproteomics -A case study from the Late Stone Age site of Toteng, Botswana

Louise Le Meillour, Séverine Zirah, Joséphine Lesur, Matthieu Lebon, David Pleurdeau, Antoine Zazzo

To cite this version:

Louise Le Meillour, Séverine Zirah, Joséphine Lesur, Matthieu Lebon, David Pleurdeau, et al.. Re- covering bone and tooth proteins in arid environments using palaeoproteomics -A case study from the Late Stone Age site of Toteng, Botswana. Scientific Symposium Frontiers in Heritage Science, Feb 2019, Paris, France. 10, pp.181 - 186, 2019, �10.13140/RG.2.2.21813.12008�. �hal-02551368�

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/331122690

Recovering bone and tooth proteins in arid environments using

palaeoproteomics - A case study from the Late Stone Age site of Toteng, Botswana

Poster · February 2019

DOI: 10.13140/RG.2.2.21813.12008

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Recovering bone and tooth proteins in arid environments using palaeoproteomics

A case study in the Late Stone Age site of Toteng, Botswana

In Africa, the absence of wild ancestors’ remains of domestic caprines indicates an exogenous origin of domestic sheep and goat. Archaeological evidences suggest that they were introduced in the Northern part of the continent by the 7^th millenium BCE, but only arrived in the Southern part by the 2^nd century BCE. Moreover, the morphological similarities existing between the two species among them, and with other small wild bovids like antelopes combined with the large amount of highly fragmented remains due to arid environnemental conditions have substantially limited the use of classical comparative anatomy analyses.

We report here the use of a promising method, palaeoproteomics, on degraded bone and tooth splinters from the Late Stone Age site of Toteng, Botswana. This study is among the first to focuse on African archaeological remains, strongly degraded by arid burying conditions and diagenesis.

The purpose of our work is to both document the presence of domestic caprines at this site, but also to develop a new methodology adapted to archaeological remains from arid environments.

a Unité Archéozoologie, Archéobotanique : Sociétés, Pratiques et Environnements (AASPE), MNHN, CNRS, Paris, France.

b Unité Molécules de Communication et Adaptations des Microorganismes (MCAM), MNHN, CNRS, Paris, France

c Unité Histoire naturelle de l’Homme Préhistorique (HNHP), MNHN, CNRS, Paris, France

* llemeillour@mnhn.fr

Louise L

^E

M

^EILLOURa,b

*, Séverine Z

^IRAHb

, Joséphine L

^ESURa

, Matthieu L

^EBONc

, David P

^LEURDEAUc

& Antoine Z

^AZZOa

Conclusion

A _bstr act

Re lts su

The advent of herding practices remains a key event in human societies in terms of social, cultural,

Co ⁿ

tex t o ^f th e P hD

In Africa, no remain of wild ancestors species of sheep Ovis aries nor goat Capra hircus has never been reported.

This suggests that those domestic species have been

The first occurences of those animals remains in the archaeological record are attested since the 7^th millenium BCE in Egypt and Lybia^[2].

But their appearance in the Southern part of the continent is only dated to the 2^nd century BCE [3].

The modalities of their diffusion accross the continent in such large

amount of time is still debated : cultural diffusion (percolation) or population migrations; Eastern or

Western route?

Regarding the strong evidences of extant exchanges between Eastern and Southern Africa, we propose to document the hypothesis

of the Eastern route using biomolecular approaches through a larger project (Fig. 1). Here, we will focus on

Aim

economical changes and regarding their interactions with animals.

introduced from the epicentre of their domestication : the Near East^[1].

Ma ter _i a l & M ^etho ds

Because of the

strong morphological

similarities between the caprines species and with other small wild

bovids, emphasized by the African burying

conditions, the distinction of archaeological remains is not always possible.

We aimed to use a novel biomolecular method for species

determination of archaeological material:

palaeoproteomics (Fig. 2).

Based on the amino acid variations on preserved proteins, this method allow species identification of remains where DNA might not be conserved, due to increased diagenetic processes amplified by arid environmental conditions.

m/z

%

Fig. 2: General principle of palaeproteomics analyses on archaeological material Archaeological remain Chemical preparation Species identification

through MS data

18 unidentified bones and teeth splinters from the Late

M

^ATERIAL

Stone Age site of Toteng, Botswana, were selected for this study. Toteng is a complex of open-air sites discovered in 1991 by A.C. Campbell where a rich sequence of typical Late

Stone Age lithic industry, a large faunal spectrum and

Bambata sherds

^[4,5,6]

were excavated. New excavations in 2003

and

zooarchaeological analyses unraveled the presence of domestic sheep (Ovis aries) directly dated to 2020 ± 40 BP (2σ, ^[7]). Remains included to this and examined by one of the authors (JL). Most of them were too fragmented and altered to propose morphological identifications beyond

family^[8].

M

^ETHODS

Fig. 1: A

rchaeolo

gical site

s include

d to the P

hD project

Estimation of remains organic phase preservation

Wavenumber (cm^-1)

Absorbance (arbitrary units)

Amide I Amide II

CO₃

ν₃PO₄ ν₄PO₄

400 900

1400 1900

2400 2900

3400 3900

In order to estimate the organic content of Toteng remains, 0.5 mg of powdered sample were applied on the diamond of a Attenuated Total Reflectance Fourier transform infra-red spectrometer. Anvil pressure was adjusted to normalise ν₃PO₄ band to an absorbance of 0.5 (Fig. 3).

The ratio between amide I and PO₄ bands area is then used to estimate nitrogen and collagen weight percent content (%wt) according to a method described by

Lebon and collaborators^[9]. Fig. 3: ATR FT-IR spectrum of a modern bone reference (B.

taurus). Areas used for organic content preservation estimation appear in grey.

Paleoproteomics

Because no chemical preparation protocol was never adapted to remains from arid environments, we aimed at identifying one that allow the best protein sequence coverage and permits species identifications (Fig. 4).

Once proteins were properly extracted and enzymatic hydrolysis succeeded, we performed high performance liquid chromatography coupled to tandem mass spectrometry analyses for characterization^[9].

(2) Decalcification 3 decalcification agents tested orange: Tris-EDTA (0.05 M & 0.5 M,

pH 7.4)

blue and green: HCl 0.6 and 1 M (1) Sampling

between 10 and 150 mg (1)

(2)

(3) Solubilisation (3) NH₄HCO₃ 50 mM Enzymatic hydrolysis

trypsin, 1:100

%

m/z

(4)

(4) UHPLC-MS/MS Fig. 4: Flow chart of palaeoproteomics protocol conducted in this study

R

^EFERENCES

[1] V^IGNE, J.-D., ^{ET AL}. , 2012, Ethnozootechnie 91, 11–19.

[2] L^ESUR, J., 2017, Presses universitaires du Midi, 205 p.

[3] P^LEURDEAU D., ^{ET AL}. 2012, PLoS ONE, 7, e40340.

A

CKNOWLEDGEMENTS

[4] C^AMPBELL A. C., 1992.

[5] R^OBBINS L., ^{ET AL}., 1998, South Afr. Arch. Bull., 53, 125-132.

[6] S^ADR K., 1997, Current Anthropology, 38, 104-112.

[7] R^OBBINS L. ^{ET AL}., 2005, Current Anthropology, 46(4), 671-677.

[8] L^E M^EILLOUR L. ^ETAL., 2018, Paleo3, 511, 472-482.

[9] L^EBON M. ^{ET AL}., 2016, Radiocarbon, 58, 131-145.

B

^{EST SUITED PROTOCOL FOR REMAINS FROM ARID}

ENVIRONMENTS

All 18 Toteng remains presented contrasted organic phase preservation. Nine out of the 18 were selected for palaeoproteomics analyses regarding a range of preservation distributed between 1.91% and 5.38%^[8] of collagen content.

TOT_03 TOT_06 TOT_07 TOT_09 TOT_11 TOT_14 TOT_15 TOT_18 TOT_26

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Tris-EDTA 0.6 M HCl 1 M HCl 49

28 32 30

7 4

94

29 23

62

20 33

Fig. 5: Number of assigned peptides using available databases per sample and per decalcification protocol used. Preserved collagen content (coll. wt%) are presented above each sample.

204.1345 333.1772 519.2409 711.3306 832.4011 925.9343

1011.4740

187.0712 300.1192 357.1404 462.2196 555.2413 687.3306 744.3524 857.3996 1140.5325

454.1923 840.3923 897.4161

200 400 600 800 1000 1200 m/z

GEPGPTGVQGPPGPAGEEGK

y₁ y₂

147.1125

y₃

y₄ y₅

y₇ y₈

y₁₈²⁺

y₉ y₁₀

954.4542

y₁₁

y₁₂ b₂

b₃ b₄ b₅

b₆

b₈ b₉ b₁₀

612.2626

b₇

[M+H]⁺

648.8070

y₁₄²⁺

y₁₄²⁺

b₈

y₁ y₂ y₃ y₄ y₅ y₈ y₉ y₁₀ y₇ y₁₁ y₁₂ y₁₈²⁺

b₂b₃b₄b₅b₆b₇ b₉b₁₀

GEPGPTGIQGPPGPAGEEGK

226.1187 283.1400 383.7324 496.2881 556.3203 669.4049 766.4571 823.4777 898.4014 955.4218

127.0865 1119.6255827.3613

303.1779 602.2532 673.2919 894.5162 1120.6271

200 400 600 800 1000 1200 m/z

b₂ - H₂O

b₆ b₇

b₈

730.3122

b9

b₁₀ b₁₁

y₃ ^412.2431

y₁₁²⁺

y₁₂²⁺

y₆

y₇ y₈

y₉

y₁₀ y₁₁ y₁₂

y₁₃

991.5665 1048.5898

524.7985

y₈²⁺

y₉²⁺

1180.5332

SGDRGEAGPAGPIGPVGAR

870.4080

a₁₀ a₁₁

927.4266

a13-NH3

435.7061

a₁₀²⁺

478.2162

b₁₁²⁺

y₁₃ b₈

y₃ y₆ y₈ y₉ y₁₀ y₇ y₁₁ y₁₂ b₂ b₆b₇ b₉b₁₀b₁₁

SGDRGETGPAGPIGPVGAR

Within the nine tested remains for palaeoproteomics analyses, four yielded preserved peptides that were assigned to proteins using available databases (Fig. 5).

Identified remaining proteins were α1 and α2 chains of collagen type I . Tris-EDTA buffer allowed the best protein sequence coverage.

129.0659 258.1089 469.7330 518.2593 596.3046 768.3887 825.4101 938.4573 1092.5304

511.2514 624.2991 681.3205 778.3737 891.4184 948.4401

147.1129 276.1559 333.1773 404.2140 501.2670 558.2886 1191.5989

200 400 600 800 1000 1200

b₂

b₃

b₄

357.1722

b₅

414.1986

b₆

b₇ b₈ b₉

b₁₀ b₁₁

1035.5093

y₁ y₂ y₃

y₄ y₅

y₆ y₇

671.3361

y₈ y₉

y₁₀

y₁₁ y₁₂ y₁₃ y₁₀²⁺

y₁₁²⁺

y₁₂²⁺

546.7701

y₁₃²⁺

AGEVGPPGPPGPAGEK

y₆ b₈

y₁ y₂ y₃ y₄ y₅ y₈ y₉ y₁₀ y₇ y₁₁ y₁₂ y₁₃

b₂b₃b₄b₅b₆b₇ b₉b₁₀b₁₁

PGEVGPPGPPGPAGEK PGEAGPPGPPGPAGEK

m/z

S

^PECIES IDENTIFICATION BASED ON PROTEIN CHARACTERISATION

Α1 chain of collagen type I allowed the refining of species identification. One remain presented the suidae marker (Fig. 6, top left), two other the equidae marker (top right) and one remain was attributed to the domestic caprine species, Ovis aries (bottom center)^[8].

All the α1 chain of collagen type I markers presented here were never reported in the litterature and will allow species identification based on the least changing chain of this type of collagen.

Fig. 6: MS/MS spectra of family and species markers of alpha 1 chain of collagen type I preserved in Toteng samples and identified in this study.

Tris-EDTA buffer is identified as the best

demineralizing agent when dealing with remains from

arid environment.

A collagen content threshold of ~ 3 wt%

under which it would be risky to perform

palaeoproteomics analyses is proposed.

confirmed based on preserved proteins

from one of the degraded splinters.

The presence of domestic sheep Ovis aries at the

Late Stone Age site of Toteng, Botswana, is

This work was supported by a grant from the ANR under the LabEx ANR-10-LABX-0003-BCDiv, in the program “Investissements d'avenir” ANR-11-IDEX-0004-02 and an ATM grant from the Muséum National d'Histoire Naturelle under the name “ProtéArch”.The authors would like to thank the National Museum of Botswana for access to the Toteng remains and expressly M. Mvimi for obtaining the exporting permit, without whom this study will not have been possible. The authors also want to thank the Plateau Technique de Spectrométrie de Masse Bio-Organique and the Plateau de Spectrométrie Infrarouge of the MNHN for accessing the instruments and X. Gallet for his help for FTIR analyses. Finally, the author would like to thank the organizers of the 8th Bone Diagenesis Meeting for provoking the opportunity of publishing in a Special Issue of the Palaeo3 journal.

methodological developments of palaeoproteomics in the context of African remains.

study were exported from the National Museum of Botswana under authorization

Leopard CaveGeduld

Las Geel Kerma

Mouweis Wakarida KumaliGaru Mota Cave

GvJm44 Kurub

Asa Koma Hedaito le Dora

Wakrita

2.57% 2.18% 3.11%

5.72%

2.94%

3.58%

5.38%

2.03% 1.91%

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