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In this work, two sets of experiments were carried out to assess the potential of soil proteomics for archaeological site interpretation. First, we examined the effects of various protein isolation reagents and soil constituents on peptide mass fingerprinting (PMF) of soil-like materials spiked with bovine serum albumin (BSA). In a subsequent case study, we assessed the relative age of soils from an ancient clay floor of a Roman farmhouse using amino acid racemization and then applied MALDI-TOF-MS-MS to detect and identify biomarkers for human occupation. The results from the first experiments indicate that BSA isolation efficiencies are hampered by the presence of clays and reactive organic matter (glucose), whereas these effects are, to some extent, reversed by the presence of humic acids. We also show that specific regions of the BSA molecule (the out most α-helical regions of domain 2 and 3) are more susceptible to isolation than other regions and this suggest that soil proteins can be only partly isolated. Soil-protein interactions were also found to inhibit tryptic cleavage of BSA, resulting in an enhanced specificity of BSA peptides. Our results further stress the importance of multiple or sequential protein isolation protocols with a focus on citrate and hydrofluoric acid. Results from the case study indicate that the ancient floor samples contain degraded and most likely aged proteinaceous matter, probably of keratinaceous origin. © 2012 Elsevier Ltd.

There is increasing evidence that certain microbially-derived compounds may account for part of the aquatic dissolved organic nitrogen (DON) pool. Enantiomeric ratios of amino acids were used to assess the microbial input to the DON pool in the Florida Everglades, USA. Elevated levels of d -alanine, d -aspartic acid, d -glutamic acid and d -serine indicated the presence of peptidoglycan in the samples. The estimated peptidoglycan contribution to amino acid nitrogen ranged from 2.8 ± 0.1% to 6.4 ± 0.9%, increasing with salinity from freshwater to coastal waters. The distribution of individual d -amino acids in the samples suggests additional inputs to DON, possibly from archaea or from abiotic racemization of l -amino acids.

We report the most comprehensive study of survival of peptide bonds and epitopes (antibody binding sites) in fossil shells from a semi-continuous New Zealand brachiopod sequence extending for 3 Ma. The study reveals for the first time long-term trends in proteins survival. The investigation focused on a sub-set of the total skeletal biomolecules, those protected from exposure to a strong oxidising agent (NaOCl); the so-called intra-crystalline component. The extent of peptide bond hydrolysis was compared with the declining immunological signal. The proportion of free amino acids increased very rapidly but between 5 and 10% of the amino acids remained peptide bound in all samples. The pattern of loss of immunological reactivity broadly mirrored the loss of peptide bonds, but overall loss of signal was much greater. Significant antibody response was observed in some but not all late Pliocene fossils (>3 Ma), but against a panel of antisera the pattern of reactivity was lost in samples >0.5 Ma. Alternative models of polypeptide chain scission were used in an to attempt to relate the rate of peptide bond hydrolysis to the loss of immunological determinants. The findings suggest that, despite early optimistic reports, the application of immunology to shell carbonates does not appear capable of extending into deep time.

In short polypeptides, l -aspartic acid (Asp) and l -asparagine (Asn) can undergo isomerization/deamidation reactions via a succinimide (Asu) intermediate resulting in the formation of d -aspartic acid. Some experimental evidence suggests that this reaction, which has been widely used in age determinations, is severely repressed in structured regions of proteins, like for example the triple-helix of collagen, indicating that conformational constraints imposed on the local peptide geometry by the peptide superstructure may inhibit the formation of Asu-containing sequences relative to Asp- or Asn-containing sequences. To examine this, molecular mechanics and molecular dynamics calculations have been performed on constrained and unconstrained Gly–Pro–Asn–Gly–Pro and Gly–Pro–Asu–Gly–Pro pentapeptides. In the constrained calculations, the influence of the protein helix is simulated by fixing the distance between the backbone-nitrogens in the first and last residue at 11.80 A, a distance similar to that between comparable atoms in the collagen triple-helix. The results from these simulations show that the constraint significantly destabilizes the Asu-containing pentapeptide relative to the Asn-containing sequence; the molecular dynamics simulations, in which the influence of water was specifically taken into account, predict that the constraint raises the energy difference between these two pentapeptides by 8.8±3.8 kcal/mol. This shows that a protein helical structure may severely repress Asp racemization and Asn deamidation. The conservation of such structures should therefore be taken into consideration when using d / l Asp ratios for age determination.

In studies of organic matter in fossil biominerals, there has been a widespread failure to distinguish between the organic matrix and organic matter trapped within the crystal elements. The existence of chemically isolated (intracrystalline) proteins are indicated by the persistence of amino acids after prolonged treatment with a strong chemical oxidant (NaOCl). The geochemical significance of these residual amino acids is illustrated by the re-analysis of aberrantly young d -aile/ l -ile ratios (0.142 ± 0.042, n = 4) of amino acids from a land snail ( Cepaea sp.) collected from Tattershall Thorpe in Lincolnshire. Following NaOCl treatment the d -aile/ l -ile ratio increased (0.178 ± 0.014, n = 5), while both the total amino acid concentration and the variance declined.