DOI: 10.5061/dryad.zkh1893d3
title: A comparison of minimally-invasive sampling techniques for ZooMS analysis of bone artifacts: MALDI-TOF mass spectra

description: Bone and antler are important raw materials for tool manufacture in many
 cultures, past and present. The modification of osseous features which
 take place during artifact manufacture frequently makes it difficult to
 identify either the bone element or the host animal, which can limit our
 understanding of the cultural, economic, and/or symbolic factors which
 influence raw material acquisition and use. While biomolecular approaches
 can provide taxonomic identifications of bone or antler artifacts, these
 methods are frequently destructive, raising concerns about invasive
 sampling of culturally-important artifacts or belongings. Collagen peptide
 mass fingerprinting (Zooarchaeology by Mass Spectrometry or ZooMS) can
 provide robust taxonomic identifications of bone and antler artifacts.
 While the ZooMS method commonly involves destructive subsampling,
 minimally-invasive sampling techniques based on the triboelectric effect
 have also been proposed. In this paper, we compare three previously
 proposed minimally-invasive sampling methods (forced bag, eraser, and
 polishing film) on an assemblage of 15 bone artifacts from the pre-contact
 site EjTa-4, a large midden complex located on Calvert Island, British
 Columbia, Canada. We compare the results of the minimally-invasive methods
 to 10 fragmentary remains sampled using the conventional destructive ZooMS
 method. We assess the reliability and effectiveness of these methods by
 comparing MALDI-TOF spectral quality, the number of diagnostic and high
 molecular weight peaks as well as the taxonomic resolution reached after
 identification. We find that coarse fiber-optic polishing films are the
 most effective of the minimally-invasive techniques compared in this
 study, and that the spectral quality produced by this minimally-invasive
 method was not significantly different from the conventional destructive
 method. Our results suggest that this minimally-invasive sampling
 technique for ZooMS can be successfully applied to culturally significant
 artifacts, providing comparable taxonomic identifications to the
 conventional, destructive ZooMS method.
description: Fifteen bone artifacts were sampled for ZooMS using three different minimally-invasive techniques: forced bag, eraser, and coarse polishing film. Sampling was conducted following modified versions of the outlined procedures in Fiddyment et al. (2015) for the eraser method (E), in McGrath et al. (2019) for forced bag method (B), and in Kirby et al. (2020) for polishing disc method (P). Sampling and extraction blanks were included for all three methods. Additionally, 10 bone fragments from Calvert Island were analyzed using the destructive acid (0.6 M HCl) demineralisation method (Buckley et al., 2009; as modified in McGrath et al., 2019). Six bone objects were tested using two different types of ultra-fine polishing film: coarse (fiber optic polishing film disc with aluminum oxide grit size of 30µm) (P1) and fine (aluminum oxide grit size 6µm) (P2).  Following individual sampling procedures, all samples were gelatinized in AmBic at 65℃ for one hour; 50µL of the resulting supernatant was removed, and the remaining pellet was stored in the freezer. Samples were incubated overnight (12–18 hours) at 37℃ with 0.4 µg of trypsin. The trypsin was deactivated using 1 µL of 5% TFA solution. Collagen in the samples was purified and desalted using Pierce C18S Tips. Each purified sample was spotted in triplicate, along with calibration standards, onto a 384-spot Bruker MALDI ground steel target plate using 1 µL of sample and 1 µL of α-cyano-hydroxycinnamic acid matrix. The samples were run on a Bruker ultraflex III MALDI TOF/TOF mass spectrometer with a Nd:YAG smart beam laser at the University of York in York, UK, and 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). Raw spectral data has been uploaded here.