DOI: 10.5061/dryad.zgmsbcch7
title: MALDI-TOF MS spectra of archaeological whale bone specimens from Atlantic Europe

description: Whale bones are regularly found during archaeological excavations.
 Identification of these specimens to taxonomic levels is problematic due
 to their fragmented state. This lack of taxonomic resolution limits
 understanding of the past spatiotemporal distributions of whale
 populations and reconstructions of early whaling activities. To overcome
 this challenge, we performed Zooarchaeology by Mass-Spectrometry on an
 unprecedented selection of 719 archaeological and palaeontological
 specimens of probable whale bone from Atlantic European contexts, from the
 Middle to Late Neolithic (c.3500–2500 BCE) to the eighteenth century CE.
 The results show high numbers of Balaenidae (most likely North Atlantic
 right whale (Eubalaena glacialis)) and grey whale (Eschrichtius robustus)
 specimens, two species no longer present in the eastern North Atlantic.
 Many of these specimens derive from contexts associated with the known
 medieval whaling cultures of the Basques, northern Spaniards, Normans,
 Flemish, Frisians, Anglo-Saxons, and Scandinavians. This association
 raises the likelihood that pre-industrial whaling impacted these taxa,
 contributing to their extinction and extirpation respectively. Much lower
 numbers of other large whale taxa were identified, suggesting that it was
 once abundant and accessible whales that suffered the greatest long-term
 impact. The pattern of natural abundance leading to over-exploitation,
 well-documented for other taxa, is thus applicable to early whaling.
description: Whale bone samples were taken
 using a ©Dremel rotary tool removing a small piece of bone weighing up to
 c.500 mg. For 474 specimens, collagen was extracted using a modified
 Longin (1971) method as detailed in Seiler et al. (2019), with the
 addition of a lipid extraction step and the use of a higher acid
 concentration, at the National Laboratory for Age Determination, Norwegian
 University of Science and Technology, Norway. 
 Initially, the samples were
 crushed into small pieces and cleaned in an ultrasonic bath with 18.2
 MΩ-cm ultrapure water (type 1) (three times five minutes). The samples
 were then ultrasonicated for 15 minutes in dichloromethane and methanol
 (2:1). This step was repeated three or more times until the solution was
 clear. Following this, the material was demineralized overnight using 2.44
 M HCl (50 ml of solution per 100 mg of bone) in glass tubes within a
 vacuum desiccator kept at room temperature. The samples were then washed
 with ultrapure water until a pH of 3 to 4 and 4 ml of 0.5 % NaOH was added
 for 2–4 hours at room temperature to dissolve humic acids. After washing
 with ultrapure water till pH&lt;10, 5 ml of 1.22 M HCl was added for
 1–2 hours to remove atmospheric CO<sub>2</sub>. The sample was
 then washed with ultrapure water till pH=3±0.2 and hydrolysed to gelatine at 70 °C
 overnight. Finally, the gelatine was filtered through a prebaked (900 °C)
 quartz filter (Merck Millipore, AQFA04700, 99.998 % capture for 0.3 μm particles) and the filtrate freeze-dried. The final
 collagen was taken to the Henry Wellcome Laboratory for Biomolecular
 Archaeology, University of Cambridge, UK, for ZooMS
 analysis.  Collagen extraction failed for 25 specimens and
 when sufficient original bone material remained a subsample was taken
 directly to the Henry Wellcome Laboratory. Additionally, another 237
 specimens were analysed by ZooMS without prior collagen extraction. For
 these 262 (25+237) specimens, approximately 30 mg subsamples were taken
 and processed at the Henry Wellcome Laboratory for Biomolecular
 Archaeology. The bones were demineralised in 0.6 M hydrochloric acid at
 4°C for two weeks. Each sample was then centrifuged, the hydrochloric acid
 discarded, and the retained material rinsed three times with 200 μl of 50
 mMol ammonium bicarbonate (AmBic) pH 8.0 solution, before being
 gelatinised in 100 μl of AmBic solution at 65˚C for one hour. For the 449
 collagen samples prepared at NTNU (474 samples, minus the 25 which
 failed), 0.1 mg of collagen was also placed in AmBic solution at 65˚C for
 one hour. For both the 262 and 449 sample groups, the
 gelatinised collagen, still in AmBic, was incubated with 0.4 μg of trypsin
 at 37˚C overnight, and subsequently acidified with 0.1% trifluoroacetic
 acid (TFA). The collagen was then purified using a 100 μl C18 resin
 ZipTip® pipette tip (EMD Millipore) with conditioning and eluting
 solutions composed of 50% acetonitrile and 0.1% TFA and washing solution
 composed of 0.1% TFA; the samples were eluted in a volume of 50 μl. Equal
 amounts of the collagen extract and α-cyano-hydroxycinnamic acid matrix
 solution (1% in conditioning solution) were mixed (1 μl each) and spotted
 onto a 384 spot MALDI target plate. Each sample was externally calibrated
 against an adjacent spot containing a mixture of six peptides
 (des-Arg1-bradykinin m/z = 904.681, angiotensin I m/z = 1295.685,
 Glu1-fibrinopeptide B m/z = 1750.677, ACTH (1–17 clip) m/z = 2093.086,
 ACTH (18–39 clip) m/z = 2465.198 and ACTH (7–38 clip) m/z = 3657.929).
 Samples were spotted in triplicate, and run on a Bruker ultraflex III
 MALDI TOF/TOF mass spectrometer with a Nd:YAG smart beam laser. A SNAP
 averaging algorithm was used to obtain monoisotopic masses (C 4.9384, N
 1.3577, O 1.4773, S 0.0417, H 7.7583). Averaged spectra were created from
 the replicates for each specimen using mMass software (Strohalm et al.,
 2008), and then visually compared to published m/z markers for mammals, as
 presented in Buckley et al. (2009), Kirby et al. (2013), Buckley et al.
 (2014) and Hufthammer et al. (2018). An additional eight specimens were
 previously analysed at BioArch at the University of York following the
 protocol outlined in Rodrigues et al. (2018) and the results incorporated
 into this study. For additional
 information see manuscript.
description: MALDI-TOF MS data was analysed using mMass software v5.5.0
 (Niedermeyer & Strohalm, 2012); details can be found in the main
 manuscript.
description: This README.txt file was generated on 2023-04-08 by Youri van den HUrk #
 Title of Dataset: MALDI-TOF MS spectra of archaeological whale bone
 specimens from Atlantic Europe Author Information Principal Investigator
 Contact Information Name: Youri van den Hurk Institution: Norwegian
 University of Science and Technology Email: yourivandenhurk@gmail.com Date
 of data collection (single date, range, approximate date): September 2021
 - March 2023 Geographic location of data collection: Trondheim, Norway;
 Cambridge, UK Information about funding sources that supported the
 collection of the data: This project is funded by the MSCA-IF project
 Demise of the Atlantic Grey whale (DAG) under the European Union’s Horizon
 2020 research and innovation programme (Grant agreement No. 101025598) and
 under the Ecology Grant of the Royal Netherlands Academy of Arts and
 Sciences (KNAW) under grant agreement KNAWWF/747/ECO2021-13. The study
 received funding from the European Research Council (ERC) Synergy Project
 4-OCEANS under the European Union’s Horizon 2020 research and innovation
 programme (Grant agreement No. 951649). One sample was analysed using
 funding received from the European Research Council (ERC) under the
 European Union's Horizon 2020 research and innovation programme under
 grant agreement No 834087 (COMMIOS). The samples from Galicia provided by
 CEMMA were collected as part of the “Galicia no Lombo da Balea II”
 project, financed by the Grupo de Acción Costeria GAC-7 Ria de Vigo-A
 Guarda. Methods for processing the data and instrument- or
 software-specific information needed to interpret the data: MALDI-TOF MS
 data was analysed using mMass software v5.5.0 (Niedermeyer &
 Strohalm, 2012); details can be found in the main manuscript ##
 Description of the data and file structure ZooMS\_Raw\_Files contains 10
 folders containing the raw ZooMS files. Each sample was ran in triplicate
 using MALDI-TOF MS. Each folder contains a plate map. The plate map shows
 which sample was spotted where and refers to the ZooMS data files within
 the folder. mMass software is required to run the data. The samples are
 numbered using a DAG number (one, two, or three digit numbers) for which
 no collagen was extracted from the sample for the MALDI-TOF MS analysis or
 a four digit number for which MALDI-TOF MS analysis was undertaken on a
 collagen sample. The numbers refer to the supplementary table submitted
 with the manuscript as a supplementary file which contains the metadata
 for each specimen (e.g. site, country, date, etc.). ## Sharing/Access
 information Links to other publicly accessible locations of the data: None
 Data was derived from the following sources: Detailed in supplementary
 table submitted with the manuscript. ## Code/Software N/A