Results
At the beginning of the project, the spectra were manually screened for
marker peaks (with a signal to noise above 10). This was extremely time
consuming. In addition, the source peak lists were all in unit mass resolution.
As the project progressed, we developed an in-house application to scan the
data for matches with a species identification list where we could specify the
signal to noise cutoff and the mass tolerance. The screening tool made the
data scanning fast and removed the potential for interpretation bias although
the data still required manual review to remove repeats of modified markers.
Methods
The methodology developed by Kirby et al
2
was used for extraction
and enzymatic digest. In brief, samples were taken by either
removing small (<1mm) loose fibres or by gently rubbing 2.5mm
diameter discs of 30 μm lapping paper over a small region (1mm
2
)
of the object. The sample was transferred to a low bind centrifuge
tube and 60 μL 50 mM ammonium bicarbonate buffer solution
added and heated at 60 °C for 45 minutes, followed by reduction
and alkylation of cysteine residues. Digestion with 8 μL trypsin
(0.02 μg/μL) was conducted overnight. Samples were stored frozen
until required for spotting on MALDI plate. Prior to spotting, the
samples were zip-tipped and eluted using the dual elution
technique into 10% acetonitrile: 90% 0.1% TFA and 50%
acetonitrile: 50% 0.1% TFA. The pH of the samples was adjusted to
2 using 10% TFA and the samples were spotted on a Fleximass-
SR48 slide. 5mg/ml CHCA matrix was used for MALDI-TOF
analysis. All peaks in the peak list were exported using
monoisotopic peak picking.
Species identification of materials used in cultural heritage objects from Alaska in the British Museum’s collection using ‘ ZooMS’ methodology
Michael Douglas Nairn
1
, Chris Mussell
2
; Amber Lincoln
2
1
Shimadzu, Manchester, UK, Manchester, United Kingdom;
2
The British Museum, London, United Kingdom
Introduction
The British Museum’s collection contains a vast number of objects
fabricated from animal derived materials where the species of
animal used is unidentified. Revealing the animal species of
worked materials provides archaeologists and curators with
insights into the past including species availability, trading networks
and technologies utilised. The information is important to
descendants of the makers who wish to understand their history
and traditions.
Initial speciation work at the British Museum via MALDI-ToF mass
spectrometry is focused on identification of species used in the
items collected from Alaska by Captain James Cook and other
explorers in the 18th and 19th centuries, including harpoons and
cordage used for hunting and fishing.
‘ZooMS’ (Zooarchaeology by Mass Spectrometry)
1-5
approach and
published marker ion libraries have been explored to facilitate
species identification.
Conclusions
Preliminary work has demonstrated the capability of the Shimadzu MALDI-
8020 instrumentation for collagen peptide mass fingerprinting and has
provided tentative identifications of animal species used in sinews found in
museum objects. Further work is required to source materials of known
taxonomic provenance to generate an in-house library, enabling complete
spectra comparison, provision of validation data and a better understanding
of intra-species variability.
Ideally a full set of species-specific marker peaks would be observed for
each sample, this has not been achievable, but is not unexpected.
Approaches for associating a confidence score to a taxonomic assignment
and presenting the data such that close, alternative, matches can be
presented alongside the most likely assignment are being investigated.
References
1. Buckley et al (2009) Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionization
time of flight mass spectrometry. Rapid Comm. Mass Spectrum. 23 3843-3854.
2. Kirby et al (2013) Identification of collagen-based materials in cultural heritage. Analyst 138 4849-4858 .
3. Kirby et al (2015) Application of a Biotechnology Technique for Accurate Identification and Regional Localization of
Mammalian Materials in Native American Cultural Heritage.
4. Buckley et al (2014) Species Identification of archaeological marine mammals using collagen fingerprinting. Journal of
Archaeological Science 41 631-641.
5. Buckley et al (2015) Corrigendum to “Species Identification of archaeological marine mammals using collagen fingerprinting”
[YJASC 41 (2014) 631-641). Journal of Archaeological Science 61 290.
Figure 2: Whale harpoon (AM.NWC.60) Sample 3.
TP-027
Figure 1: MALDI-8020 Benchtop linear mass spectrometer
Table 1: Examples of tentative species identification - based on largest number of marker
peaks observed in spectrum. Marker peaks reported as proline\hydroxyproline variants
counted as a single hit.
Figure 3: Fishing line (AM.2409)
Sample 1.
Figure 4: Sinew line (AM.VAN.143)
Sample 2.
Figure 6: Mass Spectra from fishing line sample (AM.2409) with a table of all potential
marker peaks found
Sample and location Data
Matched
peaks
1a Chugach fishing line (2019 04 30 b) Dolphin:common bottlenose white-beaked euphrosyne 8
1b Chugach fishing line (2019 04 02) Humpback whale 8
2a Sinew line (2019 01 11 a) Humpback whale 7
2a Sinew line (2019 01 11 b) Humpback whale 7
2a Sinew line (2019 04 29) Humpback whale 7
2a Sinew line (2019 04 30) Sperm whale 7
2b Sinew line (2019 01 17) Dolphin:common bottlenose white-beaked euphrosyne 7
2b Sinew line (2019 01 17) Orca/White-sided dolphin 7
2b Sinew line (2019 01 17) Porpoise 7
2b Sinew line (2019 01 17) Humpback whale 7
3a Whale harpoon (2019 04 02) Right whale 7
3a Whale harpoon (2019 04 02 b) Orca/White-sided dolphin 8
3a Whale harpoon (2019 04 02 b) Minke whale 8
3a Whale harpoon (2019 04 02 b) Fin whale 8
3a Whale harpoon (2019 04 02 b) Gray whale 8
3a Whale harpoon (2019 04 02 b) Right whale 8
3b Whale harpoon (2019 01 17) Ringed seal 7
3c Whale harpoon (2019 04 30) Dolphin:common bottlenose white-beaked euphrosyne 8
Figure 5: Spectrum obtained from caribou reference sample (relevant marker peaks
labelled with a ).
% Intensity
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
m/z
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100
2719.2
2703.0
898.2
2722.4
2707.3
2149.9
1105.4 1267.5
2687.1
1459.6
1562.8 1975.8
868.1 2134.0 2293.0
1435.6 1804.9
1182.5
3001.2 1586.7
1088.4
1166.5 1580.7 2088.9 1473.6 3017.2
840.1 2410.0
908.3 3084.3 2676.2 2776.3 1150.5 1832.8 1328.5 2883.3
1706.8 1095.3 3021.5 2215.9
2558.1
851.2
2118.0 1192.6 1847.8 955.3 1648.8 1332.6 3100.3 1261.5 1113.4 1997.9 2350.1 3036.4
2555.0 2501.1
2052.0
1763.9
1562.8 1428.0 2763.1 2705.1 2171.2 2883.2 2284.0 898.3 2651.2 1928.0 1178.4 851.0
Caribou reference sample
Chugach fishing line sample
1 2
3
% Intensity
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
m/z
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100
2375.8 2074.8
2133.7
2149.8
2241.8
2702.9
1435.6
2090.9
2718.9 1201.5 1459.6
1784.8
2359.8 898.4
1989.8 2118.8
1808.8
1267.6 1079.4
2433.8 2058.9 1478.6
1800.8
1577.7 2688.0 2798.9 2294.9 1736.7
1177.4
2352.8 2548.9
1652.7
2482.0 2190.8
1104.3 2246.7 1453.7 2132.8 1779.7 1352.6 2759.9
2067.8 2857.0 1869.7 920.3 1189.4 1258.4 2675.9
3034.0 3101.0 1067.3 1122.4
1453.7 1562.7 1990.0 1808.9 1645.7 2059.8 2145.6 1905.0 2295.6 2951.5 2525.1 1385.9 2235.3 2457.7 1715.0 2607.2 2884.3 991.2 1324.7 2763.1 2375.2
Observed m/z S/N
1079.4 217
1205.6 42
1453.7 55
1566.7 42
1638.5 16
2118.8 107
2767.6 14
2866.1 12
