Using chiral amino acid analysis by reverse-phase high pressure liquid chromatography (RP-HPLC), we assessed the rate of amino acid degradation during thermal degradation of modern chicken feathers heated dry in modern Wollemia nobilis resin. Despite a slew of recent papers investigating protein fossilisation3,4,28,29 protein degradation is still not fully understood, and this is especially true in the unusual chemical environment found in amber. The experiments therefore give us a framework to understand the patterns of amino acid racemisation, and changes in amino acid composition, during fossilisation in amber. These experimentally aged feathers in resin showed very low levels of protein degradation, with D/L < 0.5 for all amino acids in all samples, even after 504 hours at 140 °C (Fig. 2, Table S3), comparable to or slightly lower than the levels of protein degradation seen in feathers thermally degraded in air (Fig. S1, Tables S4 and S5). Significantly, this is considerably slower than rates of protein degradation in comparable experiments on other sample types which contained closed system protein, including ostrich eggshell28, coral29, and mollusc shells30 (Fig. 3). The modern chicken feathers in resin also showed a decrease in concentration (Table S2) and a continuous increase in racemisation of amino acids with increased heating time and increased temperature (Fig. 2).

Figure 2 Amino acid analyses. (A) Amino acid compositions and D/L values for the taphonomic experiments and fossil analyses. (B,C) Comparisons of the amino acid compositions of taphonomic experiments and fossil analyses (this paper) to dust49, human sweat50,51, and human fingerprints/fingertips33,34,52, using only the amino acids Asx, Glx, Ser, Thr, His, Arg, Ala, Val, Leu, Ile. These 10 amino acids were the only ones for which previously-published data was provided for common amino acid contaminants; the compositions from our experimental and analytical samples were recalculated using only these ten amino acids. (B) Principal component analysis (PCA). (C) Amino acid compositions. Full size image

Figure 3 Mean D/L values of Asx, Glx, and Val during thermal maturation experiments of modern samples, including Gallus g. domesticus feathers in resin (this paper), ostrich eggshell28, limpet shell30, and coral29. (A) 110 °C. (B) 140 °C. Note that the other experiments (ostrich eggshell, limpet shell, and coral) included environmental water, whereas the experiments reported in this paper (feathers in resin) did not include any environmental water. Full size image

We also investigated nine fossil specimens (ranging from 44 Ma to 105 Ma in age, Supplemental Table S1, Fig. 1). In seven cases, we could not detect amino acids, suggesting that in these samples all the protein had degraded. Two samples, one each from Eocene (~44 Ma) Baltic amber (Fig. 1A)31 and Cenomanian (~99 Ma) Burmese amber (Fig. 1E)32, had detectable amino acids; of these two samples, we recovered lower concentrations of amino acids from the older Burmese amber feather than from the younger Baltic amber feather (Supplemental Table S2). The primary goal in analysing these amino acids is to rigorously test the null hypothesis that they represent contamination rather than ancient, endogenous, amino acids.

The sampling and analytical methods were carefully developed to prevent introducing modern contamination during our handling of the specimens. The lack of detectable amino acids in seven of our nine samples suggests that this was successful; if modern contamination was introduced during sampling, all samples would likely have been affected.

Although we limited the introduction of contamination during our sampling, modern contamination could also have been introduced by others between the time of collection and analysis. In this case, we would expect the composition of the amino acids to be more similar to a contaminant than to feathers. Human keratin would be the most likely contaminant; previous studies have indicated that contamination of fossils with human keratin primarily occurs through direct skin contact (e.g. the fingerprint/fingertip amino acids) or through keratin dust as part of laboratory dust33,34. The proteins present in the fingerprints/fingertips and sweat are primarily human keratin33,35; these both provide a direct test of human keratin contamination. Laboratory dust is a mixture of human keratin and proteins from other sources33,34; including multiple samples of laboratory dust provides a range of comparative mixtures. Other possible contaminants can be introduced during cleaning fossils, keratin from animal hair brushes used to clean fossils or protein-containing glue used to repair fossils; however any brushes would have only made contact with the outer surface of the amber, which we removed prior to analysis, and these samples were not glued. The amino acid composition of the feathers (both fresh and experimentally degraded in resin) does not overlap with any of the contaminant samples including human keratin (Fig. 2), indicating that contamination can be distinguished from feather amino acids. Both fossil amino acid samples are more similar to modern chicken feathers degraded in resin than to dust, fingerprints/fingertips, or sweat, a few of the most likely modern contaminants (Fig. 2), suggesting these amino acids were indeed from the enclosed fossil feather. The Baltic amber sample specifically falls exactly along the line of experimental feather degradation in resin (Fig. 2), indicating that the composition is exactly what we would expect for a feather in resin that is slightly more degraded than in our experiments. The Burmese amber sample does not so clearly match feathers degraded in resin, but it falls between fresh feathers and feathers degraded in resin, and is not similar to any contaminant (Fig. 2).

More evidence that at least some of this signal was from ancient amino acids came from the pattern of amino acid racemisation, which can indicate whether amino acids are modern or ancient. The degree of racemisation in some hydrophobic amino acids, such as Tyr, Val, Phe, Leu, Ile (particularly in the Baltic amber sample), is much higher than would be expected, or indeed than would be possible, for modern contamination (Supplemental Table S3, Fig. 2). The fossils have undergone much more extensive decay than the experimental samples, and should therefore have higher D/L values. This is the case for the high D/L values of the hydrophobic amino acids (e.g. Tyr, Val, Phe, Leu, Ile) recovered from the two fossil feathers (Table S3, Fig. 2), which strongly suggests they are (at least in large part) ancient. In contrast, the D/L values of the faster racemising amino acids (e.g. Asx and Ser) are comparatively low, inconsistent with the data from apolar amino acids, and from the experiments; we would typically expect fossils to exhibit high degrees of racemisation of these amino acids, and to preferentially lose polar amino acids as well as the most unstable amino acids28,30,36,37. The Burmese amber sample shows less racemisation than the Baltic amber sample (Fig. 2, Table S3), which is unexpected given that Burmese amber (~99 Ma) is approximately twice as old as Baltic amber (~44 Ma). This could be explained by some contamination, or by the degradation of the free amino acids themselves, which would preferentially remove right-handed amino acids from the samples. Significant contamination, however, is unlikely due to the strong feather signal in the amino acid composition. Moreover, other researchers have found that keratin proteins preserve even into the Jurassic27, suggesting that Cretaceous keratin preservation is not at all unexpected or unreasonable.

The low concentrations of amino acids in all amber samples imply that amber-entombed feathers have undergone significant diagenesis. However the variable levels of recovered amino acids further support the idea that not all fossils are equal; that two of nine samples included detectable levels is encouraging. CT and synchrotron images of amber-entombed insects sometimes demonstrate loss of the internal tissue38, suggesting that decay proceeds rapidly following entombment when there is still sufficient entrapped water to enable decay.

As amino acids from the Burmese amber feather (~99 Ma) are less racemised than the amino acids from the younger Baltic amber sample (~44 Ma), this suggests that the methods of entrapment result in multiple independent diagenetic histories. A number of variables have the potential to affect protein degradation rate. For example, the distinct resin chemistries of different ambers are indicative of preservation fidelity; some resins are characterised by almost perfect preservation, others by poor preservation38. This bias will act on proteins alongside other tissue compositions. In particular, fruit flies decay very quickly in Pinus resin (Pinaceae) and very slowly in Wollemia resin (Araucariaceae)39. Baltic and Burmese amber are chemically distinct31,40,41,42. Burmese amber is produced by an Araucareacean, phylogenetically close to Wollemia42, and although the botanic source of Baltic amber is disputed31, a number of authors have noted it likely belongs to the Pinaceae family31,43. It is possible that the decreased racemisation of the older Burmese amber, compared with the Baltic amber, could reflect a reduced decay rate due to variations in the amber chemical microenvironment and therefore its antimicrobial effects44.

In conclusion, the combination of careful sampling procedures, amino acid composition data, and amino acid racemisation data suggests that we found potentially ancient, endogenous amino acids from fossil feathers in amber. The amino acids we recovered were similar to those previously identified from insects in Baltic amber14: both showed low amino acid concentration, significant degrees of racemisation, and the presence of some reactive, polar amino acids. Furthermore, the amino acid composition of the fossils was similar to the amino acid composition of chicken feathers thermally aged in modern resin. However, the D/L values, which were higher in the younger fossil feather than in the older fossil feather, and the high levels of some polar, reactive amino acid in the fossils are not exactly what we would expect based on the experiments. The process of protein fossilisation and degradation in amber requires further study to determine if these discrepancies are due to partial amino acid contamination, or whether the experiments (3 weeks of thermal aging) do not encompass all possible pathways of chemical change and degradation in amber.