ZooMS

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Zooarchaeology by mass spectrometry, commonly referred to by the abbreviation ZooMS, is a scientific method that identifies animal species by means of characteristic peptide sequences in the protein collagen. ZooMS is the most common archaeological application of peptide mass fingerprinting (PMF) and can be used for species identification of bones, teeth, skin and antler. It is commonly used to identify objects that cannot be identified morphologically. In an archaeological context this usually means that the object is too fragmented or that it has been shaped into an artefact. Archaeologists use these species identification to study among others past environments, diet and raw material selection for the production of tools.

Contents

Developmental history

ZooMS was first published in 2009 [1] by a team of researchers from the University of York, but the term was coined later in a publication in 2010. [2] The original aim of ZooMS was to distinguish between sheep and goat. The bones of these two closely related species are difficult to distinguish, especially when fragmented, yet the difference between these two common domesticates is very important for our understanding of past husbandry practices.

Most of the method development following the initial publication of ZooMS has focused on the extraction of collagen from the archaeological material. In the original protocol acid was used to dissolve the bone’s mineral matrix and free up the collagen. In 2011 an alternative extraction method was published that used an ammonium bicarbonate buffer to solubilise the collagen without dissolving the mineral matrix. [3] In contrast to the acid protocol, the ammonium bicarbonate protocol does not affect the size and mass of the sample, making it a much less destructive method compared to the original protocol. In fact, the ammonium bicarbonate protocol was proposed as a non-destructive protocol for ZooMS, but in practice destructive samples are still taken for this protocol (see [4] ). Submerging a sample in ammonium bicarbonate does chemically alter the sample, which is why current practices continue to take a destructive sample.

Non-destructive sampling protocols

Although the ammonium bicarbonate protocol should not be considered a non-destructive method, it was followed by more ‘true’ non-destructive methods. The first of these was the eraser protocol, first tested on parchment, [5] but later also applied to bone. [6] The eraser protocol is performed by rubbing a PVC eraser on a piece of parchment or bone. The friction generates triboelectric forces, which causes small particles of the sample to cling to the eraser waste. From the eraser waste collagen can then be extracted and analysed. The eraser protocol was found to work relatively well for parchment, but it is less effective on bone. Additionally, it leaves microscopic traces on the bone surface, which appear very similar to use wear traces and could be an issue for use wear analysis. [6]

A second non-destructive protocol is the plastic bag protocol, first published in 2019. [7] It is based on the idea that the normal friction between an object and the plastic bags, commonly used for storing archaeological objects, might be sufficient to extract enough material for ZooMS analysis.

A third protocol uses the same triboelectric principle. However, instead of using an eraser, this microgrid protocol employs a fine polishing film to remove very small amounts of material from a sample. [8]

The last non-destructive protocol that has been published for ZooMS is the membrane box protocol. [9] The membrane box protocol is based on contact electrification, which is the generation of electrostatic forces due to small localised differences in charge between two objects. These electrostatic forces can be large enough for material transfer between two surfaces. [10]

Most of these protocols have only been published recently and their respective advantages and disadvantages have not yet been tested against each other. It is therefore not yet clear how reliable these methods are and what level of preservation of the samples is required for them to work.

Reference biomarkers

Apart from non-destructive sampling, a second area of method development has been the expansion of reference biomarkers. To identify a species using ZooMS, a set of diagnostic biomarkers is used. These biomarkers correspond to particular fragments of the species’ collagen protein. The set of known biomarkers at the time of ZooMS’ original publication was relatively limited, but recent publications have been expanding this list. A regularly updated list of published biomarkers is maintained by the University of York and can be found here.

Principle of the method

Fig. 1 Schematic overview of a typical ZooMS workflow ZooMS Schematic Diagram.jpg
Fig. 1 Schematic overview of a typical ZooMS workflow

ZooMS identifies species based on differences in the amino acid composition of the collagen protein. The amino acid sequence of a species’ collagen protein is determined by its DNA and as a result like DNA, the amino acid sequence reflects a species’ evolutionary history. The greater the evolutionary distance between two species, the more different their collagen proteins will be. ZooMS typically can identify a sample up to genus level, though in some cases the identification can be more or less specific. A good understanding of the archaeological context of the sample can be used to further refine the resolution of the species identification.

Protocol example

A ZooMS protocol (Fig. 1) typically consists of an extraction, denaturation, digestion and filtration step, followed by mass spectrometric analysis. Various destructive and non-destructive extraction protocols have already been discussed in some detail above. The key is to extract the protein preserved in the sample and then bring it into solution, usually an ammonium bicarbonate buffer. Denaturation is done to unfold the proteins and make them more accessible for the enzymatic digestion. It is done by heating the solubilised sample at around 65 °C. [3] Then an enzyme, trypsin, is added to the solution. Trypsin cleaves the protein after every arginine or lysine amino acid in its sequence, resulting in peptide fragments of predictable masses. After digestion the sample is filtered with C18 filters to get rid of non-proteinaceous material and the sample is now ready for mass spectrometric analysis, which for ZooMS generally means MALDI-TOF MS.

Related Research Articles

<span class="mw-page-title-main">Zooarchaeology</span> Archaeological sub-discipline

Zooarchaeology or archaeozoology merges the disciplines of zoology and archaeology, focusing on the analysis of animal remains within archaeological sites. This field, managed by specialists known as zooarchaeologists or faunal analysts, examines remnants such as bones, shells, hair, chitin, scales, hides, and proteins, such as DNA, to derive insights into historical human-animal interactions and environmental conditions. While bones and shells tend to be relatively more preserved in archaeological contexts, the survival of faunal remains is generally infrequent. The degradation or fragmentation of faunal remains presents challenges in the accurate analysis and interpretation of data.

<span class="mw-page-title-main">Proteomics</span> Large-scale study of proteins

Proteomics is the large-scale study of proteins. Proteins are vital macromolecules of all living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

<span class="mw-page-title-main">Tandem mass spectrometry</span> Type of mass spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more stages of analysis using one or more mass analyzer are performed with an additional reaction step in between these analyses to increase their abilities to analyse chemical samples. A common use of tandem MS is the analysis of biomolecules, such as proteins and peptides.

<span class="mw-page-title-main">Protein sequencing</span> Sequencing of amino acid arrangement in a protein

Protein sequencing is the practical process of determining the amino acid sequence of all or part of a protein or peptide. This may serve to identify the protein or characterize its post-translational modifications. Typically, partial sequencing of a protein provides sufficient information to identify it with reference to databases of protein sequences derived from the conceptual translation of genes.

<span class="mw-page-title-main">Peptide mass fingerprinting</span> Analytical technique for protein identification

Peptide mass fingerprinting (PMF), also known as protein fingerprinting, is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. The method was developed in 1993 by several groups independently. The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match.

<span class="mw-page-title-main">Matrix-assisted laser desorption/ionization</span> Ionization technique

In mass spectrometry, matrix-assisted laser desorption/ionization (MALDI) is an ionization technique that uses a laser energy-absorbing matrix to create ions from large molecules with minimal fragmentation. It has been applied to the analysis of biomolecules and various organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. It is similar in character to electrospray ionization (ESI) in that both techniques are relatively soft ways of obtaining ions of large molecules in the gas phase, though MALDI typically produces far fewer multi-charged ions.

Protein methods are the techniques used to study proteins. There are experimental methods for studying proteins. Computational methods typically use computer programs to analyze proteins. However, many experimental methods require computational analysis of the raw data.

<span class="mw-page-title-main">Deamidation</span> Chemical reaction

Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine or glutamine is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid. Glutamine is converted to glutamic acid or pyroglutamic acid (5-oxoproline). In a protein or peptide, these reactions are important because they may alter its structure, stability or function and may lead to protein degradation. The net chemical change is the addition of a water group and removal of an ammonia group, which corresponds to a +1 (0.98402) Da mass increase. Although deamidation occurs on glutamine, glycosylated asparagine and other amides, these are negligible under typical proteolysis conditions.

Surface-enhanced laser desorption/ionization (SELDI) is a soft ionization method in mass spectrometry (MS) used for the analysis of protein mixtures. It is a variation of matrix-assisted laser desorption/ionization (MALDI). In MALDI, the sample is mixed with a matrix material and applied to a metal plate before irradiation by a laser, whereas in SELDI, proteins of interest in a sample become bound to a surface before MS analysis. The sample surface is a key component in the purification, desorption, and ionization of the sample. SELDI is typically used with time-of-flight (TOF) mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples, however, SELDI technology can potentially be used in any application by simply modifying the sample surface.

Phosphoproteomics is a branch of proteomics that identifies, catalogs, and characterizes proteins containing a phosphate group as a posttranslational modification. Phosphorylation is a key reversible modification that regulates protein function, subcellular localization, complex formation, degradation of proteins and therefore cell signaling networks. With all of these modification results, it is estimated that between 30–65% of all proteins may be phosphorylated, some multiple times. Based on statistical estimates from many datasets, 230,000, 156,000 and 40,000 phosphorylation sites should exist in human, mouse, and yeast, respectively.

<span class="mw-page-title-main">Thermospray</span>

Thermospray is a soft ionization source by which a solvent flow of liquid sample passes through a very thin heated column to become a spray of fine liquid droplets. As a form of atmospheric pressure ionization in mass spectrometry these droplets are then ionized via a low-current discharge electrode to create a solvent ion plasma. A repeller then directs these charged particles through the skimmer and acceleration region to introduce the aerosolized sample to a mass spectrometer. It is particularly useful in liquid chromatography-mass spectrometry (LC-MS).

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<span class="mw-page-title-main">Protein mass spectrometry</span> Application of mass spectrometry

Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.

<span class="mw-page-title-main">Top-down proteomics</span>

Top-down proteomics is a method of protein identification that either uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry (MS/MS) analysis or other protein purification methods such as two-dimensional gel electrophoresis in conjunction with MS/MS. Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. The name is derived from the similar approach to DNA sequencing. During mass spectrometry intact proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance, quadrupole ion trap or Orbitrap mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry (MS). Top-down MS (non-gel) proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.

<span class="mw-page-title-main">Quantitative proteomics</span> Analytical chemistry technique

Quantitative proteomics is an analytical chemistry technique for determining the amount of proteins in a sample. The methods for protein identification are identical to those used in general proteomics, but include quantification as an additional dimension. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about the physiological differences between two biological samples. For example, this approach can be used to compare samples from healthy and diseased patients. Quantitative proteomics is mainly performed by two-dimensional gel electrophoresis (2-DE), preparative native PAGE, or mass spectrometry (MS). However, a recent developed method of quantitative dot blot (QDB) analysis is able to measure both the absolute and relative quantity of an individual proteins in the sample in high throughput format, thus open a new direction for proteomic research. In contrast to 2-DE, which requires MS for the downstream protein identification, MS technology can identify and quantify the changes.

The in-gel digestion step is a part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced in 1992 by Rosenfeld. Innumerable modifications and improvements in the basic elements of the procedure remain.

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<span class="mw-page-title-main">Matthew Collins (academic)</span>

Matthew Collins, is a professor at the University of Copenhagen, formerly as a Niels Bohr professor, and also holds a McDonald Chair in Palaeoproteomics at the University of Cambridge.

Stable isotope standards and capture by anti-peptide antibodies (SISCAPA) is a mass spectrometry method for measuring the amount of a protein in a biological sample.

<span class="mw-page-title-main">Ancient protein</span>

Ancient proteins are complex mixtures and the term palaeoproteomics is used to characterise the study of proteomes in the past. Ancients proteins have been recovered from a wide range of archaeological materials, including bones, teeth, eggshells, leathers, parchments, ceramics, painting binders and well-preserved soft tissues like gut intestines. These preserved proteins have provided valuable information about taxonomic identification, evolution history (phylogeny), diet, health, disease, technology and social dynamics in the past.

References

  1. ↑ Buckley, Michael; Collins, Matthew; Thomas-Oates, Jane; Wilson, Julie C. (2009-12-15). "Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: Species identification of bone collagen using MALDI-TOF-MS". Rapid Communications in Mass Spectrometry. 23 (23): 3843–3854. doi:10.1002/rcm.4316. PMID   19899187.
  2. ↑ Buckley, M., S. W. Kansa, S. Howard, S. Campbell, J. Thomas-Oates & M. J. Collins. 2010. Distinguishing between archaeological sheep and goat bones using a single collagen peptide. Journal of Archaeological Science 37: 13-20.
  3. 1 2 van Doorn, Nienke Laura; Hollund, Hege; Collins, Matthew J. (2011-09-01). "A novel and non-destructive approach for ZooMS analysis: ammonium bicarbonate buffer extraction". Archaeological and Anthropological Sciences. 3 (3): 281–289. doi:10.1007/s12520-011-0067-y. ISSN   1866-9565. S2CID   85056079.
  4. ↑ Naihui, Wang; Samantha, Brown; Peter, Ditchfield; Sandra, Hebestreit; Maxim, Kozilikin; Sindy, Luu; Oshan, Wedage; Stefano, Grimaldi; Michael, Chazan; Liora, Horwitz Kolska; Matthew, Spriggs; Glenn, Summerhayes; Michael, Shunkov; Kristine, Richter Korzow; Katerina, Douka (2021-02-20). "Testing the efficacy and comparability of ZooMS protocols on archaeological bone". Journal of Proteomics. 233: 104078. doi:10.1016/j.jprot.2020.104078. ISSN   1874-3919. PMID   33338688. S2CID   229325462.
  5. ↑ Fiddyment, Sarah; Holsinger, Bruce; Ruzzier, Chiara; Devine, Alexander; Binois, Annelise; Albarella, Umberto; Fischer, Roman; Nichols, Emma; Curtis, Antoinette; Cheese, Edward; Teasdale, Matthew D.; Checkley-Scott, Caroline; Milner, Stephen J.; Rudy, Kathryn M.; Johnson, Eric J. (2015-12-08). "Animal origin of 13th-century uterine vellum revealed using noninvasive peptide fingerprinting". Proceedings of the National Academy of Sciences. 112 (49): 15066–15071. doi: 10.1073/pnas.1512264112 . ISSN   0027-8424. PMC   4679014 . PMID   26598667.
  6. 1 2 Sinet-Mathiot, Virginie; Martisius, Naomi L.; Schulz-Kornas, Ellen; van Casteren, Adam; Tsanova, Tsenka R.; Sirakov, Nikolay; Spasov, Rosen; Welker, Frido; Smith, Geoff M.; Hublin, Jean-Jacques (2021-12-08). "The effect of eraser sampling for proteomic analysis on Palaeolithic bone surface microtopography". Scientific Reports. 11 (1): 23611. doi:10.1038/s41598-021-02823-w. ISSN   2045-2322. PMC   8655045 . PMID   34880290.
  7. ↑ McGrath, Krista; Rowsell, Keri; Gates St-Pierre, Christian; Tedder, Andrew; Foody, George; Roberts, Carolynne; Speller, Camilla; Collins, Matthew (2019-07-30). "Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points". Scientific Reports. 9 (1): 11027. doi:10.1038/s41598-019-47299-x. ISSN   2045-2322. PMC   6667708 . PMID   31363122.
  8. ↑ Kirby, Daniel P.; Manick, Annette; Newman, Richard (2020-10-01). "Minimally Invasive Sampling of Surface Coatings for Protein Identification by Peptide Mass Fingerprinting: A Case Study with Photographs". Journal of the American Institute for Conservation. 59 (3–4): 235–245. doi:10.1080/01971360.2019.1656446. ISSN   0197-1360. S2CID   210522155.
  9. ↑ Martisius, Naomi L.; Welker, Frido; Dogandžić, Tamara; Grote, Mark N.; Rendu, William; Sinet-Mathiot, Virginie; Wilcke, Arndt; McPherron, Shannon J. P.; Soressi, Marie; Steele, Teresa E. (2020-05-08). "Non-destructive ZooMS identification reveals strategic bone tool raw material selection by Neandertals". Scientific Reports. 10 (1): 7746. doi:10.1038/s41598-020-64358-w. ISSN   2045-2322. PMC   7210944 . PMID   32385291.
  10. ↑ Galembeck, Fernando; Burgo, Thiago A. L.; Balestrin, Lia B. S.; Gouveia, Rubia F.; Silva, Cristiane A.; Galembeck, André (2014-11-24). "Friction, tribochemistry and triboelectricity: recent progress and perspectives". RSC Advances. 4 (109): 64280–64298. doi:10.1039/C4RA09604E. ISSN   2046-2069.
  11. ↑ Brown, Samantha; Douka, Katerina; Collins, Matthew J; Richter, Kristine Korzow (2021-03-20). "On the standardization of ZooMS nomenclature". Journal of Proteomics. 235: 104041. doi:10.1016/j.jprot.2020.104041. ISSN   1874-3919. PMID   33160104. S2CID   226279979.
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