RESULTS

The 11 archeological samples analyzed ranged in age from 40 years to approximately 50,000 years (Table 1); 10 represent mineralized tissues (bone and tooth) from a variety of environmental conditions with one soft tissue remain from the Siberian permafrost. In addition, one sample of modern human epidermis was analyzed for comparison. Py-GC/MS was performed on all 12 samples. Fig. 1 shows the resultant chromatograms, and Tables 2 and 3 summarize the results.

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Figure 1

Total ion chromatograms of pyrolysates of the 11 samples studied. (A) Fresh skin; (B) Mammuthus primigenius; (C) Equus caballus; (D) Mylodon darwinii; (E) Equus ferus; (F) Equus hemionus; (G) Homo neandertalensis; (H) Papio cf. cynocephalus; (I) Ursus spelaeus; (J) Equus ferus; (K) vertebrate; (L) Megalonyx sp. Numbers and symbols above the peaks correspond to Table 2. ∗, contaminants; ⋅, nonextractable lipids; B_n, alkylbenzenes; I_n, alkylindenes; N_n, alkylnaphthalenes; F_n, fluorene; n, the extent of alkyl substitution (i.e., 0, none; 1, methyl; 2, dimethyl or ethyl; etc.).

Two samples yielded few or no pyrolysis products that could be assigned to biological molecules. One (Table 2, Megalonyx sp.) was a 13,000-year-old tooth from Florida that showed a series of alkylated aromatic compounds such as benzenes, indenes, naphthalenes, and fluorenes (Fig. 1L). Previously, similar products have been seen in pyrolysates of fossil arthropod cuticles (17). The origin of these compounds may be either the result of extensive diagenetic alteration of the endogenous macromolecules or an artifact of the pyrolysis procedure (32). Alternatively, such compounds may stem from contamination of the sample; such contamination would agree with the racemization results, as a D/L ala ratio greater than the D/L asp ratio is known to be a marker for contamination (33). The second sample (Table 2, Vertebrate) was a 60,000-year-old bone from South Africa that showed only traces of two pyrolysis products related to proline (Fig. 1K). The other nine samples contained a complex mixture of compounds. The large majority of these pyrolysis products can be assigned to amino acids and protein debris either by virtue of their molecular structures or by comparative interpretation of pyrolysates of polypeptides, collagen, and fresh skin. For example, pyrroline (no. 1; numbers refer to those in Fig. 1), pyrrole (no. 2), C1 (no. 4), and C2 (no. 6) alkylpyrroles are characteristic products of proline; diketodipyrrole (no. 15) and pyrroline (no. 1) are derived from hydroxyproline, whereas toluene (no. 3), styrene (no. 5), ethylcyanobenzene (no. 7), and propylcyanobenzene (no. 10) are produced during the pyrolysis of phenylalanine (23, 24). Furthermore, several 2,5-diketopiperazines are seen. These are formed via a so-called “McLafferty rearrangement” (23, 34, 35) during pyrolysis of dipeptide sequences in which proline is one of the amino acids. In most of the samples studied, the following products can be assigned to 2,5-diketopiperazines, which can be recognized in terms of their dipeptide precursors: Pro-Ala (no. 14), Pro-Gly (no. 15), Pro-Val and/or Pro-Arg (no. 17), Pro-Hyp (no. 18), and Pro-Pro (no. 19).

The fact that derivatives of proline, glycine, alanine, and hydroxyproline are prominent in the pyrolysates suggests that collagen may be present in the samples. Collagen, the most abundant protein in mammalian skin and bone, consists of three polypeptide chains in which glycine (≈33%), alanine (≈10%), proline (≈12%), and hydroxyproline (≈10%) are the major constituents. To investigate whether other products seen in the pyrolysates may be derived from collagen, a sample of collagen and fresh skin (Fig. 1A) was pyrolyzed in parallel with the ancient tissues. Besides many minor pyrolysis products, compounds no. 8 and no. 9 (tentatively identified as a piperidinone derivative), alkylpyrimidones (no. 11 and no. 12), and a pyrazine derivative (no. 13) were all prominent in the collagen, fresh skin, and some of the ancient samples. Thus, a majority of the organic compounds detected in the pyrolysates seem to be assignable to collagen-type debris. Indeed, in agreement, relatively large amounts of products assignable to hydroxyproline are also detected in the samples.

Four of the samples also contained C₁₆ and C₁₈ fatty acids, n-alk-1-enes and n-alkanes, alkyl-nitriles, and amides, presumably derived from bound lipid components (Fig. 1 C, D, F, and G, indicated by ⋅ symbols). The specimen of the bone from a 40-year-old horse showed the highest relative abundance of the latter components (Fig. 1C). These lipids may be bound to tissue structures or encapsulated in micropores such that they cannot be removed completely by organic extraction before pyrolysis.

Table 2 summarizes the relative amounts of pyrolysis products (highest peak assigned as 100) related to single amino acids and 2,5-diketopiperazines on all samples. Table 3 gives the ratio of amino acid peak height to 2,5-diketopiperazine peak height, the extent of racemization of two amino acids (3, 29), the extent of oxidative DNA damage to the pyrimidine bases (5-hydroxy-5-methylhydantoin and 5-hydroxyhydantoin) assessed by GC/MS (2), and the length of DNA fragment amplifiable from the extract. It has been possible to retrieve DNA sequences from six of the samples. These positive results have been verified through reproduction within a single laboratory and, with the sole exception of the 40-year-old sample, by workers in other laboratories (2–7, 29–31). It has proven impossible to amplify DNA sequences from the remaining five samples, despite repeated attempts with various extraction protocols all performed in the same laboratory.

DISCUSSION

Of the 11 samples analyzed, samples B–H (as identified in Fig. 1 and Table 2) show good levels of protein debris as indicated by the relative amounts of pyrolysis products compared with those of collagen and fresh skin, especially those products assigned to diketopiperazines. Samples I and J had fewer relative amounts of diketopiperazines, whereas samples K and L lacked any indication of protein preservation.

There is no clear correlation between the relative abundance of most pyrolysis products assigned to single amino acids (e.g., peaks 1–7) and the preservation of endogenous DNA fragments. For example, the relative abundance of these products is similar in samples E and I, but the latter has not yielded genuine ancient DNA sequences. However, the relative abundance of the products assigned to 2,5-diketopiperazines in the pyrolysate of samples B–H is greater than those of samples I–L. Additionally, one peak, no. 17 (Pro-Arg and/or Pro-Val), was not detectable in samples I–L. The preservation of peptide linkages involving these amino acids does show a better correlation with DNA preservation. In fact, by looking at the ratio of the summed peak heights of single amino acid products and the summed peak heights of the 2,5-diketopiperazine products (Table 3, AA/DKP), we observed a distinct hierarchy of preservation. For samples B–H, the ratio of AA/DKP peak heights ranged from 2–10, with fresh skin tissue at 5; however, samples I and J both had ratios of 40 and 41. We interpret this ratio as suggesting the extent of hydrolysis of the intact proteins within the sample. In cases in which preservation has been good and little hydrolysis has taken place, the peptides are longer in length, and, therefore, more diketopiperazines are formed due to the pyrolysis procedure. On the other hand, when peptides are cleaved into shorter fragments because of a more complete hydrolysis, the pyrolysis of these tissues yields fewer diketopiperazines. Therefore, this ratio may be one way to estimate the relative amounts of peptide hydrolysis in the samples.

Proteins hydrolyze under geochemical conditions through cleavage of an internal peptide bond, an internal amino lysis reaction at the N terminal position, and hydrolysis at the C terminal position (36). In bones, the hydrolysis of collagen is rapid, and little intact collagen is found after 10,000–30,000 years in temperate environments (36).

Of the seven samples (B–H) that show good protein debris, the first six have yielded authentic DNA sequences (Table 3). In addition, five of these samples have D/L ratios for aspartic acid between 0.05 and 0.07, whereas the Neanderthal-type specimen had a D/L ratio for aspartic acid of 0.12. The remaining samples that failed to yield DNA sequences had D/L ratios of 0.15 or higher and have between 2.7–20.2 and 5.3–29.2 times more oxidized pyrimidine bases than those samples that yielded DNA sequences. Thus, in cases where protein preservation is good and little racemization and base oxidation has taken place, amplifiable DNA is present. The Py-GC/MS results of the Neanderthal-type specimen indicate good protein preservation with relatively high levels of diketopiperazines. These results agree with the amino acid racemization and DNA results published for this specimen (29). Nevertheless, the baboon bone (sample H) shows that these different parameters of preservation do not always correlate well with each other. This sample yielded relative amounts of all pyrolysis products comparable to those of samples C and D but failed to yield any authentic ancient DNA. Additionally, the ratio of amino acids to diketopiperazines was 7 and thus well within the range (2–10) of “good” samples. Thus, the preservation of proteinaceous debris in bone alone does not guarantee the presence of amplifiable DNA. Despite these results, the sample did have relatively high amounts of oxidatively damaged bases and a moderately high level of racemization (D/L Asp of 0.18).

It is generally believed that the degradation of organic matter in fossils is a function of limited microbial activity (37, 38). Environments where bacterial growth is restricted, such as permafrost, hypersaline pools, acidic moorlands, and arid regions, may therefore provide good long-term preservation. However, although such environments often ensure excellent morphological preservation, they are not always conducive to macromolecular preservation. A case in point may be the Egyptian baboon bone; it is well preserved macroscopically, and protein debris is relatively well preserved as assessed by Py-GC/MS. The bone seems to have been protected from extensive microbial degradation, a situation typical with many remains from Egypt because of the arid climate (39). In spite of this preservation, the sample does not yield amplifiable DNA, which is in fact typical of most naturally and artificially mummified remains of humans and animals from Egypt (40). Therefore, the recognition of Py-GC/MS products compatible with a collagen origin does not guarantee DNA survival, a result which is in agreement with previous results (3). It is possible that samples preserved under these circumstances have undergone condensation or Maillard-type reactions, which may have cross-linked the proteins with the DNA, and thus the samples may contain endogenous DNA that is inaccessible to current methods of DNA extraction (as has been shown recently in coprolites from the Southwestern United States; ref. 41). Further work is needed to elucidate this possibility. Additionally, samples deposited in warm environments may be preserved from extensive hydrolysis through rapid drying but are still subject to oxidative damage, which would block the extension of the polymerase in the PCR. Samples like these are lost sources of ancient DNA unless the DNA can be repaired before DNA amplification.

The results presented here are restricted to relatively few samples, because the number of ancient specimens from which the authenticity of ancient DNA sequences have been rigorously established is limited. Nevertheless, the results indicate that Py-GC/MS may provide a fast and sensitive way to differentiate samples that are unlikely to yield any DNA from those that may contain DNA. At present, both Py-GC/MS and amino acid analyses that allow the extent of racemization to be determined seem suited for the purpose of identifying samples suitable for DNA extraction (3, 42). Additionally, Py-GC/MS has the advantage of being able to determine the extent of diagenetic alteration of a sample more easily than other methods of analysis on fossil material. On the other hand, Py-GC/MS is a less sensitive method than HPLC coupled with fluorescence and can yield products that are often the result of rearrangements of the original molecules occurring during the pyrolysis procedure. Recent analysis of amber-entombed fossils by Py-GC/MS (43) has shown extensive diagenetic alteration contrary to the results obtained from an amino acid analysis by HPLC (44), which indicated amino acid preservation and low racemization. It may be that these particular amino acids were in low concentration and thus not detectable by Py-GC/MS; in addition, they may have been “masked” by the resin markers in the total pyrolysate. Based on the diagenetic transformation of more decay-resistant molecules such as lignin and chitin, it was postulated that more decay-prone molecules such as DNA would not survive (43). In any event, fossils that are millions of years old will require even more discriminatory methods of analysis before the exact nature of preservation can be elucidated. As the number of ancient samples from which verified DNA sequences have been published increases, it will be possible to evaluate more precisely which of these methods of analysis best correlates with DNA preservation. In any event, because the amplification of DNA by the PCR is notoriously prone to contamination, it is extremely valuable to analyze protein preservation from archaeological and paleontological remains before DNA analysis.

Our knowledge of the influence of particular environmental conditions on the preservation of biomacromolecules is still incomplete. In spite of this fact, most investigations stress that a favorable burial environment will limit biodegradation and thus enhance preservation. However, preservation is often selective, and in some cases, the presence of one molecule will not necessarily guarantee the persistence of another. In general, preservation seems to follow a predicted hierarchy of molecules dictated by the relative strength of their bonds (45). This model, however, does not take into consideration the fact that molecules may be altered once they are cross-linked, adsorbed to minerals, and/or trapped within glycosylation end products, resulting in preferential preservation. Although permafrost may provide an excellent burial condition for biomacromolecules, it is still not clear which environmental factors may enhance preservation of some macromolecules, while, at the same time, leading to the degradation of others. In any event, time is not the crucial factor when the specimens are deposited/buried in a favorable environment where the initial rapid degradation can be inhibited.