The use of proteomics in analysing archaeological human remains is growing rapidly – used to determine biological sex, age-at-death, and even health. As this area expands, however, so does our realisation of what we still don’t know about the technology and its applications. For instance, it has largely been taken for granted that proteins are distributed uniformly throughout teeth – or at least equally across their layers, as teeth form in childhood in ways not dissimilar to tree rings. This appears to be evident, for instance, in cases of hypoplasia where malnutrition or illness causes noticeable bands across the teeth where the enamel is depleted, which coincide with periods of stress at the time that that part of the tooth was forming. No research has looked into whether this assumption is correct, however. To rectify this, Joannes Dekker, along with researchers from the Universities of York, Copenhagen, Chester, and Leiden, used a type of mass spectrometry called matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) to visualise the two-dimensional distribution of proteins in both archaeological and modern teeth. In this month’s ‘Science Notes’, we explore the results from this project.
The study sampled nine archaeological teeth, which had been obtained from an assemblage excavated from the Eusebius Church graveyard in Arnhem, the Netherlands (c.1350-1829), and four modern teeth, which were donated after they had been removed for non-pathological reasons. For consistency, the team aimed to select apparently healthy teeth, primarily premolars, from men aged 18-35. After demineralising the teeth to soften them, they were then cut into micro-thin sections, approximately 13μm thick. These samples then underwent MALDI-MSI, which created an image showing how a particular target mass was distributed across the surface of the section. Images of masses associated with different peptides from the same protein were then combined to show the 2D distribution of the selected protein throughout the tooth.
While there are more than 800 proteins present in the human tooth, the team focused for this project on the four that are of most interest archaeologically. These were collagen type I alpha-1 and alpha-2, two forms of the same protein, which are frequently used to identify the species the tooth came from as well as for radiocarbon dating and isotope analysis; alpha-2-HS-glycoprotein, which is used to determine age-at-death; haemoglobin subunit alpha, which indicates the presence of blood; and myosin light polypeptide 6, which was selected as a control as it is primarily found in muscle tissue and is not common in bones or teeth.
The results showed that these four proteins were not evenly distributed across all of the teeth, whether modern or archaeological, nor even within each individual tooth. (The image – ABOVE – shows the distribution of the four proteins in two of the analysed teeth.) Instead, while some proteins were more homogenously distributed, others seemed to have specific ‘hot spots’ where the protein was concentrated. The cause of these ‘hot spots’, however, remains a mystery, as they do not seem to align with either the growth lines of teeth or with the function of a specific protein (for instance, the centre of the tooth, the dental pulp, contains blood vessels and it would be expected that the haemoglobin protein might be found in larger quantities around the pulp cavity, but this was not the case). Even more unexpectedly, despite being part of the same collagen triple helix, and hence part of the same molecule, collagen alpha-1 and collagen alpha-2 did not have the same distribution pattern.
Summarising their conclusions in a paper recently published in Rapid Communications in Mass Spectrometry (https://doi.org/ 10.1002/rcm.9486), the team state that ‘several implications are clear. It cannot be assumed that any single sample of a tooth accurately represents the relative protein abundance within the whole tooth. In addition, the fact that protein distribution does not appear to follow the growth pattern of teeth raises doubts regarding the possibility of correlating intratooth differences in protein abundance or composition with life history events, without further work to refine the MSI methods presented in this study.’
While these conclusions might be seen as suggesting the pilot study created more questions than it answered, what it has shown is that MALDI-MSI has a lot of promise for helping us to visualise protein distribution better. It has also exposed several areas for future work, which could lead to us gaining a better understanding of why these proteins have variable distributions and whether these patterns can be predicted. If so, this would mean that we could avoid unnecessary destructive sampling in the future by focusing on the part of the tooth that is most likely to yield the protein of interest.