Proteomic profile of bone "collagen" extracted for stable isotopes: Implications for bulk and single amino acid analyses.

RATIONALE: Protein studies in archaeology and paleontology have been dominated by stable isotope studies to understand diet and trophic levels, but recent applications of proteomic techniques have resulted in a more complete understanding of protein diagenesis than stable isotopes alone. In stable isotope analyses, samples are retained or discarded based on their properties. Proteomics can directly determine what proteins are present within the sample and may be able to allow previously discarded samples to be analyzed. METHODS: Protein samples that had been previously analyzed for stable isotopes, including those with marginal and poor sample quality, were characterized by liquid chromatography/mass spectrometry using an LTQ Orbitrap Velos mass spectrometer after separation on a Dionex Ultimate 3000 LC system. Data were analyzed using MetaMorpheus and custom R scripts. RESULTS: We found a variety of proteins in addition to collagen, although collagen I was found in the majority of the samples (most samples >80%). We also found a positive correlation between total deamidation and wt% N, suggesting that deamidation may impact the overall nitrogen signal in bulk analyses. The amino acid profiles of samples, including those of marginal or poor stable isotope quality, reflect the expected collagen I percentages, allowing their use in single amino acid stable isotope analyses. CONCLUSIONS: All the samples regardless of quality were found to have high concentrations of collagen I, making interpretations of dietary routing based on collagen I reasonably valid. The amino acid profiles on the marginal and poor samples reflect an expected collagen I profile and allow these samples to be recovered for single amino acid analyses.

The effects of burning on isotope ratio values in modern bone: Importance of experimental design for forensic applications.

This study examined preservation of isotope ratio values by comparing isotope composition of bones before and after burning. We analyzed common geoprofiling isotope systems (δ13C, δ15N, δ18O, and 87Sr/86Sr) and lesser studied systems (δ34S and δ88/86Sr) to evaluate if inferences about diet and residence history were altered by the burning process. We used two burn methods: one to simulate previous academic studies using a muffle furnace and one to more closely resemble a house fire or body disposal attempt using open flame. To mimic previous burn studies, ribs and femora from four dry modern human skeletons were heated in a muffle furnace. To resemble a forensic burn situation, fleshed pig ribs from a single geographic location were burned on an open fire both with and without use of a diesel accelerant. Isotope ratios from bone collagen, carbonate, phosphate, and strontium were analyzed. Fleshed pig samples burned in an open fire maintained unaltered isotope ratio values. Dry human samples burned in a muffle furnace maintained unaltered isotope ratio values in most isotope systems, except for δ18O values in carbonate and phosphate, which showed a depletion of 18O at higher temperatures. This research suggests that the isotope composition of fleshed burned bone retains the geoprofiling inferences of unburned bone, at least within the parameters of the open fire burn used in this study. However, oxygen isotopes of carbonate and phosphate from dry bone burned in a muffle furnace do not retain the geoprofiling inferences. This research demonstrates the need for research using an experimental design relevant to a specific burn situation.