Investigating the relationship between glutamine deamidation and collagen breakdown in bone

Bone can survive in the burial environment for millions of years and can provide direct information about individuals, during their lifetime and post mortem. However it is not currently possible to directly date bone material beyond the limits of radiocarbon dating ~50,000 years before present(BP). It was suggested by van Doorn et al., (2012), that deamidation in bone collagen may be related to thermal age. However, a number of bones investigated from the same site resulted in a large range of glutamine deamidation measurements. This thesis therefore explores in detail the potential of glutamine deadmidation as a dating tool for bones older than 50,000 yeas BP. The work presented in this thesis investigates a number of potential causes for the observed variability. It was found that the pre-treatment of bone with HCl not only increased levels of deamidation, but also caused significant levels of swelling and breakdown of the collagen fibril structure. Areas of bone exhibiting localised macroscopic degradation were shown to be correlated with an increase in the levels of glutamine deamidation. This indicates that the measurement of deamidation may be used as a method by which to investigate the preservation state of bone. The analysis of synthetic peptide mixtures, containing glutamine or glutamic acid showed that an ionisation bias occurs when using MALDI, with product peptide E consistently underestimated. However, analysis of the same peptide mixtures using ESI showed no ionisation bias between the two peptide products and is therefore preferable for the measurement of glutamine containing peptides. Kinetic heating experiments performed at high temperatures (80 ᵒC -140 ᵒC), have proven unsuitable for the measurement of deamidation within the buffer soluble collagen fraction. Deamidation in the buffer soluble collagen fraction was successfully measured at low temperature (65 ᵒC), and resulted in an increase in deamidation over time. However levels of deamidation in the mineral bound fraction extracted from the same bone chips heated at 65 ᵒC did not appear to undergo deamidation over time; indicating that glutamine deamidation in this fraction is slower and therefore may be suitable for the analysis of preservation of older bone from the Late Pleistocene Period. Levels of deamidation in bones analysed from nine sites ranging in age from ~ 8000 - 30,000 years BP showed a correlation with levels of glutamine deamidation and thermal age. However, using the current methodology, it is not possible to identify the age of the sites using only the measured levels of deamidation. An adapted extraction method has been suggested to further investigate the relationship between deamidation and the thermal age of bone. The work carried out in this thesis has directly contributed to the broader knowledge of how collagen degrades in bone over time, and thus has wide reaching implications within the archaeological scientific community.