Morphology.
As portrayed in Fig. 2a ~ e, the animal hairs had different morphological characteristics, including thickness, length, density, etc. Except for lamb, the hairs of the other four animals had dense underhairs and fewer guard hairs. The hair of the grey fox (Fig. 2a), which was short and straight, had a complex color pattern. As shown in Fig. 2b, the raccoon dog’s guard hairs were up to twice as long as the underhairs, while the underhairs were much denser than guard hairs. The hair of the American mink (Fig. 2c) had a shiny appearance because a large number of the short guard hairs reflect light. The hair of the cape hare (Fig. 2d) was dense, soft, and short, making it warm, while the hair of the lamb (Fig. 2e) was long and curly.
As previously described, the animal hairs were of 2 types, and in this paper, the underhairs were chosen to observe their scales because they account for most of the hair. The longitudinal features of the five animal hairs were quite different, as shown in Table S1 and Fig. 2f ~ j. The grey fox hair fibres, as shown in Fig. 2f, had tile-shaped scales, whereas the raccoon dog hair (Fig. 2g) and American mink hair (Fig. 2h) had thin strip scales. The most notable hair was cape hare hair (Fig. 2i), as the scales of the cape hare hair had a unique herringbone shape. The thickest hair fibre among the five animals belonged to the lamb, which was approximately 30 µm, while the other 4 animal hairs were between 10–19 µm. The scales of the lamb hair were ring-shaped, as shown in Fig. 2j. The scales of hair from raccoon dogs and American minks were similar to each other, which may be because they are both from the order carnivora. A similar observation was made in the measurement of the longitudinal features, as shown in Table S1, where both the height of the scale and the ratio of diameter to scale height (d/l) from raccoon dogs and American minks were similar. Despite the similar scale structures, it is still not difficult to distinguish between them in the measurement of the longitudinal features. The scale density of the hairs from raccoon dogs was approximately 51.84/mm, while that from American minks was approximately 80.33/mm. Among the five animals, cape hare hair had the maximum scale density (up to 185/mm), which may be attributed to the herringbone-shaped scales. In addition, lamb hair had a maximum d/l ratio of 2.46. The closer the value of d/l is to 1, the closer the rectangular scale is to a perfect square; thus, the scales of the cape hare hair had the flattest ring shape.
The characteristics of the cross sections of the animal hairs are exhibited in Fig. 2k ~ o and Table S2. All five animal hairs had medullary cavities. Except for lamb (Fig. 2o), the distribution of cross-sectional morphologies from the other 4 animal hairs was uneven because they all have underhairs and guard hairs. For medullary cavities, the cross sections of cape hare hair (Fig. 2n) were so notable because it had not only single columns but also double columns, plurality of columns and no medullary cavity. The morphology of the cross sections of grey fox hair (Fig. 2k) was close to a circle, and we found the same result in Table S2. The ratio of the major axis to the minor axis (a/b) of grey fox hair was 1.35, which is close to 1. Raccoon dog hair (Fig. 2l) had two types of cross sections: one was close to a circle, similar to the grey fox hair, and the other was oval; thus, the value of a/b was 1.68 and higher than that of the grey fox hair. However, the grey fox still had a similar major axis, minor axis and cross-sectional area in the hair cross sections compared with raccoon dogs. Grey foxes and raccoon dogs are both Canidae, and they are carnivores along with American minks. Although the morphology of the cross sections from American mink (Fig. 2m) was not like those of the grey fox or raccoon dog, the American mink had a similar a/b value compared with the raccoon dog. Moreover, the measurement of cross-sectional morphologies in cape hare hair was divided into two parts: some with at most one column medullary cavity and the other with multiple column medullary cavities. Despite the cape hare, the major axis, minor axis and cross-sectional area in the lamb were the maximum among the animals studied, which was consistent with the size of the animal bodies. In short, we established the morphological database of five species of animal hairs, and we found that this method was effective for original samples that had not been woven, dyed, or aged, and was especially suitable for species that were not similar in kinship. However, if it is a historical object that has lost morphological characteristics, this method may become difficult to perform and not be accurate enough.
Molecular Structure.
FTIR and XRD are well-established techniques that can yield information on the molecular conformation of animal hairs that cannot be detected by morphological methods. Both of these techniques are applied to analyse the differences in animal hairs at the molecular level. The FTIR results were shown in Figure S1a, five animal hairs exhibited similar spectra, which were consistent with the structural characteristics of the keratin. In order to distinguish the five animal hairs, secondary structures were analysed through FTIR spectra. As depicted in Figure S1b, different species were distinguished except grey fox and raccoon dogs, both of which are canines. The XRD patterns of all animal hairs were indistinguishable, conforming to the structural features of keratin, as illustrated in Figure S1g. And the secondary structure could not be obtained by the XRD through peak fitting due to the overlap of α-helix and β-sheet diffraction bands. Consequently, species identification of animal hairs cannot be achieved by XRD method.
Molecular weight.
SDS-PAGE is a well-established and widely accepted experimental method for segregating proteins and analysing protein molecular weight. As shown in Figure S2a, there were no distinct differences in the staining patterns for keratin in each lane. Although the colour depth of each lane was diverse, this might owing to the colour of the animal hairs themselves, and it could not be the evidence identifying the five species of animals.
Species Identification of Hair Textiles via Paleoproteomics.
To further investigate the hair textiles, LC-MS/MS was performed to analyse the species source of the keratin extracted from the five animal hairs. The base peak chromatograms of the samples are shown in Fig. 3a. Canine samples of grey fox and raccoon dogs exhibited similarities in their chromatograms, implicating the similarity of the protein structure between these two samples before enzymatic hydrolysis. For the five animals (grey fox, raccoon dog, American mink, cape hare, lamb), the database of mammalian keratin was searched, as shown in Fig. 3b ~ c, 88, 128, 64, 145, 102 proteins and 286, 206, 228, 323, 138 peptides were detected, respectively. In order to get more in-depth information about species identification, the intensities of the top ten proteins of the five species are displayed in Table S3 with the exclusion of mismatched proteins from animals that cannot be used in textiles such as Gorilla gorilla gorillas and Tursiops truncatus. In grey fox hair, A0A3Q7RA57 and A0A3Q7SLK7 had high scores and their intensities were identified as components of keratin originating from Vulpes vulpes, the animal closest to the grey fox in the UniProt database. Keratin, type II cuticular Hb1 isoform X2 (A0A2Y9KMQ0; A0A2Y9KNE8) and keratin, type II cuticular Hb6 (A0A2Y9KVB6) derived from Enhydra lutris kenyoni were detected, which may be because of the hybridization of Vulpes vulpes with the Enhydra lutris kenyoni genome. Interestingly, A0A3Q7RA57, A0A3Q7SLK7 and A0A2Y9KVB6 were also detected in American mink hairs. This could be attributed to the fact that both grey fox and American mink are from the order Carnivora. This phenomenon also occurred in cape hare hair. A0A2Y9KPT5 and A0A2Y9K0E9, ascribed to Enhydra lutris kenyoni and G9KSS7 and U6CX15 ascribed to Mustela putorius furo were found, whereas G1SEN0 ascribed to Oryctolagus cuniculus was detected with low scores. This was because the database of Leporidae keratins is extremely inadequate compared with other mammalian species of keratins in the UniProt database and the score depended on the database, making the score of G1SEN0 lower. In raccoon dog hair, F1P922 derived from Canis lupus familiaris was detected, while E3VW87 and P02441 derived from Ovis aries were detected only in lamb hair. Among them, E3VW87 with more unique peptides could be selected as a biomarker for discerning hair produced by Ovis aries from the other four animals.
To obtain biomarkers of the other four species, the four samples were cross-identified against the Canidae, Neovison vison and Leporidae databases (Table S4) for more precise keratin information. In cape hare hair, only G1SEN0 and G1SUH8 were detected, and G1SUH8 had more Razor + unique peptides; thus, G1SUH8 was selected as a biomarker. Both U6CZM3 and U6DQL8, with many unique peptides, existed only in American mink hair, meaning that both could be established as biomarkers. However, cross-reactivities were found when searching against the Canidae database, and keratins in more than one species are not suitable as biomarkers. Finally, A0A3Q7RKS7 with 5 unique peptides was used as the biomarker of grey fox hair, while Q6EIZ1 and F1PTX4 with 3 unique peptides were established as biomarkers of raccoon dog hair. Thus, we obtained the biomarkers of the five animal hairs. By searching these biomarkers in the proteomic results, the animal sources of hair textiles will be determined.
Analyses of Ancient Hair Textiles.
The ancient hair samples shown in Fig. 1 were provided by the China National Silk Museum. Sample A was unearthed from Xiaohe cemetery (1800 BC), and samples B and C were unearthed from Shanpula cemetery (217 BC-283AD). The methods established above on the modern samples were performed for the identification of ancient samples, and the morphological results are displayed in Fig. 4a and Table S5. The digital photos (Fig. 1) show that the length and thickness of all the hair tended to be the same, unlike those animals with many guard hairs and underhairs. The scales of the three samples recorded by SEM (Fig. 4a) were all ring-shaped; however, the surface of the fibres was seriously corroded and some scales were fallen off, which may affect the measurements to some extent. The measurements of the fibres are illustrated in Table S5. The fibres of three samples were thicker than grey fox, American mink and cape hare, but close to lamb. The height of the scale was also close to that of the lamb but partially overlapped with that of the grey fox and cape hare. The density of scale was close to the American mink and lamb; nevertheless, observation of the density of scale was affected by corrosion on fibres, and therefore could not be accepted, and neither could the ratio of d/l. In conclusion, the morphological results of the three samples are closer to those of wool. Slicing requires a large amount of fibre, and XRD requires samples with a large weight; therefore, both of these methods are not suitable for the identification of ancient samples. The molecular structure and molecular weight distributions of ancient samples were analyzed by infrared spectra and SDS-PAGE, respectively. As observed in Figure S1c, the ancient samples were identified to be protein absolutely, however, the species sources of the ancient hair textiles could not be determined via secondary structure analysis, especially for the larger proportion of random conformation than that of the five modern samples (Figure S1d), which was due to the destruction of ordered structure after thousands of years of burial. As depicted by Figure S2b, the molecular weight distributions of sample C was between 15–100 kDa, which was narrower than sample A and sample B of 15–250 kDa. This indicated the more severe aging of sample C, consistent with the analysis results of the secondary structure.
Unlike inferring from the structure, LC-MS/MS was introduced to determine the species of samples based on the peptides present. The three ancient samples showed similarity in their base peak chromatograms, as displayed in Fig. 4b, indicating that the proteins were similar, and the three samples might be derived from the same species. The database of mammalian keratin was searched for three ancient samples, as shown in Fig. 4c ~ d, 65, 89, 100 proteins and 263, 298, 319 peptides were detected, respectively. The number of proteins and peptides in ancient samples was comparable to that in modern samples, while conventional methods were influenced by the impurities and degradation of ancient samples, indicating that the paleoproteomics method is more stable and more suitable for ancient samples. The proteomic results of three ancient samples are shown in Table S6. All of the biomarkers were obtained before were searched against the proteomic results, and only E3VW87 of Ovis aries was found in the three ancient samples, as shown in Table 1. The identified peptides of E3VW87 in lamb hair and ancient samples are shown in Tables S13 ~ S16. A total of 9, 8, and 8 species-diagnostic peptides of E3VW87 in lamb hair were detected in sample A, sample B and sample C, respectively. Therefore, the analysis results revealed that the species sources of all three ancient samples were sheep, verifying the conclusion of the morphological method.
Table 1
Proteins bearing species-diagnostic peptides identified in ancient hair textiles.
Sample
|
Protein IDs
|
Razor +
unique peptides
|
Unique
peptides
|
Score
|
Intensity
|
Sample A
|
E3VW87
|
3
|
3
|
30.37
|
439330000
|
Sample B
|
E3VW87
|
2
|
2
|
20.832
|
175500000
|
Sample C
|
E3VW87
|
3
|
3
|
5.9541
|
93439000
|
In order to further validate the identification results of ancient samples, the species-diagnostic peptides of sheep were selected through the following steps. First, the LC-MS/MS results of lamb and other four modern animal samples were searched against the corresponding species Uniprot databases, and then the unique peptides in different species were analysed by Venn diagram, as shown in Fig. 5a, the lamb possessed 93 unique peptides. Among the 93 unique peptides, the peptides that may exist in the other 4 species were screened out by blasting analysis, while the peptides that only exist in wool keratin were retained. Finally, a total of 7 reliable sheep species-diagnostic peptides were selected for the ancient samples verification, as displayed in Table S7, three peptides marked with an asterisk exist in biomarker E3VW87. The final identification results were shown in Fig. 5b, 5, 3, and 4 sheep species-diagnostic peptides were detected in sample A, B, and C, respectively. The sheep species-diagnostic peptide AHYDDIASR existing in protein biomarker E3VW87 were detected in all three ancient samples, and the peptides YEEEIALR existing in E3VW87 were detected both in sample A and sample C, proving the correctness of the previous paleoproteomics identification results via protein biomarkers.