DOI: 10.25338/b8w644
title: Using bone technology and ZooMS to understand indigenous use of marine mammals at Iita, northwest Greenland

description: The site of Iita in northwestern Greenland had near-continual occupation
 from 1000 CE to the mid-20th century, which presents an unparalleled
 opportunity to examine diachronic changes in the use of marine mammals for
 food and raw material resources by Pre-Inuit (Late Dorset) and
 Inuit-Inughuit—two culturally distinct Indigenous groups. Limited wood in
 High Arctic environments necessitated the use of antler, bone, and ivory
 for tool making. Choices in the selection and processing of osseous
 material reflect changes in the way these two groups used the same
 resources. However, modification of osseous material during the
 tool-making process is often to such an extent that few diagnostic
 features remain for species identification. Here, we include
 Zooarchaeology by Mass Spectrometry (ZooMS) to identify typically
 indeterminate pieces of manufacturing debris from preserved collagen. The
 minimally destructive extraction technique using polishing films had a 99%
 success rate. The results suggest that the selection of raw materials from
 certain marine mammals, particularly narwhal, by toolmakers at Iita was
 more frequent than previously understood through traditional faunal
 analysis alone. There is a paucity of narwhal from the Pre-Inuit (Late
 Dorset) assemblage, which contrasts starkly with the high frequency of
 narwhal in the Inuit-Inughuit assemblage. We demonstrate the importance of
 combining the analysis of bone-tool manufacturing debris with traditional
 dietary faunal remains to improve our understanding of resource use in
 coastal environments.
description: One hundred and thirty specimens
 were chosen for peptide mass fingerprinting (PMF) to identify previously
 unknown osseous materials and their taxonomic origin. Identification of
 these specimens promotes a better understanding of osseous tool
 production. PMF involves the enzymatic digestion of proteins followed by
 Matrix Assisted Laser Desorption-Ionization Time of Flight Mass
 Spectrometry (MALDI) analysis of the resultant peptide mixture. In the
 case of bone and ivory, Type 1 collagen is the major constitutive protein,
 and for each mammalian source, the amino acid sequence of Type 1 collagen,
 albeit highly conserved, may have one or more amino acid substitutions.
 These substitutions change the mass of some peptides detected in the MALDI
 spectrum of the protein digest, which are then used as markers for
 specific mammalian sources. Markers from known reference materials are
 compared with those from unknown samples for identification. 
 The ZooMS (PMF) method was a
 simplified procedure based on that described in Kirby (2013, 2019). Since
 there are no cystines in collagen, the cystine reduction and end capping
 steps were omitted. In addition, spectral quality was sufficient to
 provide confident identifications in all cases where collagen was
 detected, so no purification or fractionation was required. Some spectra
 (~15%) had weak or absent A and/or G markers, but those markers were not
 required for accurate identifications of these samples. In all cases, only
 a single sample was digested and analyzed. The
 first author sampled the specimens at the UC Davis Zooarchaeology Lab with
 sample sticks—polystyrene strips with fiber optic polishing film attached
 on two sides—which are used to abrade and entrap a small amount of
 material for analysis. A freshly laundered lab coat, KN95 or surgical
 facemask, and nitrile examination gloves were worn during handling of all
 osseous materials. To avoid cross-contamination, gloves were changed, and
 the workstation was thoroughly sterilized with 70% alcohol wipes between
 each sample. Samples were obtained by rubbing the object lightly in a
 circular motion for 10 seconds. After sampling, the tip of the sample
 stick containing the abrasive film and entrapped sample is cut off, placed
 in an Eppendorf tube, and forwarded for analysis. Polishing films (30um
 alumina or diamond particles, final size 2.5 X 2.5mm) were from Precision
 Fiber Products, Inc., www.precisionfiberproducts.com. Polystyrene strips (1.5 X 2.5 X 400mm) were from Walthers, www.walthers.com. Photographs of each specimen were taken with a DinoLite digital microscope to document the area sampled. The sampling site is generally invisible to the naked eye, and the procedure consumes a miniscule amount of each specimen. Thus, it is considered a minimally destructive sampling technique by most museum curators and conservators. Each specimen and Eppendorf tube was given a unique sample number. Additional samples from the same location were collected on three specimens (KNK912x300; KNK3930x1571, x1822) because they yielded low-quality spectra on the first sample run. These are considered “second passes”, in which collagen was sampled from the same location but from a deeper anatomical component of the specimen, such that a better quality and/or greater quantity of collagen could be obtained. PMF analysis of the 130 samples collected was then undertaken at the Northeastern University Mass Spectrometry Core Facility.  Digestion: 60µL of 50mM ammonium bicarbonate (AMBI) were added to each sample (on the sample stick tip in a 600µL Eppendorf tube) and heated to 75°C for 60min. After cooling, 8µL Promega Sequence Grade trypsin (0.02µg/µL in 50mM AMBI) was added and digestion proceeded overnight at 37°C.  MALDI analysis: 2µL of the digest were added to 20µL 40% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) with saturated α-Cyano-4-hydroxycinnamic acid (CHCA) matrix. 0.65µL of the mixture was spotted onto the MALDI plate. Spectra were obtained with an Applied Biosystems/Sciex 5800 MALDI-TOF-TOF instrument operated in positive reflector mode. Calibration was done with a standard mixture of peptides: 757.3992Da, 1046.5418Da, 1296.6848Da, 1347.7354Da, 1619.8223Da, 2093.0867Da, 2465.1983Da, and 3147.4710Da. Spectra were coadditions of 1200 – 2000 laser shots. Acquired spectra were exported from Applied Biosystems Data Explorer software as text files and imported into mMass (Strohalm et al. 2010) for analysis. Spectra were manually inspected for markers. Markers used to identify mammalian sources are compiled from Buckley et al. (2009, 2014) and Kirby (2013).
description: Data is provided in two formats, .msd and .txt files. The text
 files are organized so that they correspond to the data in Supplementary
 Table 1. Further details can be found in the README.md file.
 The .txt files can be accessed by various software programs to
 visualize the data.  The .msd files can be viewed using
 the mMass software for those who have access. Alternatively, the .msd
 files can be read by using the packages MALDIQuant and MALDIQuantForeign
 in R.  <strong>mMass Data Miner: an Open Source
 Alternative for Mass Spectrometric Data
 Analysis</strong><br>Strohalm M, Hassman M, Košata B, Kodíček
 M<br><em>Rapid Commun Mass Spec</em> 22 (6), 905-908
 (2008)<br>DOI: 10.1002/rcm.3444