Located in the middle of Henan Province, China, Jiahu is one of the most representative early Neolithic Age ruins in central China. Twenty ¹⁴C dates indicate that the settlement developed over three sub-periods: 9,000 BP to 8,500 BP, 8,500 BP to 8,000 BP, and 8,000 BP to7,500 BP [1–2] (no isotope dates have been obtained directly from the tombs; however, they should fall into these three categories). The site is famous for the discovery of the earliest playable musical instrument (bone flutes) [2], the earliest mixed fermented beverage of rice, honey and fruit [3], the earliest domesticated rice in northern China [4], and possibly the earliest Chinese pictographic writing [5]. The excavated biological remains, including pollen, phytoliths and soil micromorphology, indicate that Jiahu’s warm and humid climate not only favoured the growth of mulberry trees, which feed the silkworm, but also enabled Jiahu inhabitants to settle and develop agriculture [6].

Evidence indicates that the earliest clothes made from animal skin were produced approximately 70,000 years ago or more [7]. Wild flax fibres were made into textiles approximately 30,000 years ago [8]. As a unique material, silk was not used to produce textiles until a much later time; the first use of silk textile is estimated to be only 5,000 years ago [9]. Although previous findings have provided important evidence of silk-making activities, such as figuline spinning wheels and osseous lamellas with traces of filament winding [10], a lack of direct evidence remains a challenge for demonstrating the existence of silk (derived from silkworm) during the Neolithic Age. Silk fibre is a polymer composed of sericin and fibroin, which are two types of proteins. Sericin comprises a series of globular proteins that are unstable and can be damaged rapidly after long-term degradation [11–13]. Fibroin contains highly ordered structural entities that are aggregated by intra- and intermolecular hydrogen bonds. A light chain (approximately 26 kDa) and a heavy chain (approximately 390 kDa) are the two subunits that constitute the fibroin. A heavy chain molecule was identified with twelve domains from the crystalline regions, that containing several Gly-X repeats, with X being Ala, Thr, Ser or Val. A thermodynamically stable structure that ensures the heavy chain’s resistance to water, mild acidity or alkalinity and other degradation factors, was generated due to the strong hydrogen bonds and Van der Waals forces in the crystalline regions [10–17]. Whereas the light chain only connected to the heavy chain by a few disulfide bonds, and is an independent sub-unit that shows less stable properties, for instance more hydrophilic character, higher degradation rate and water uptake ability [17–19]. Hence silk-based cultural relics cannot be easily preserved in their original shape.

Much effort has been made to overcome these difficulties. Credible peptide data obtained in our previous studies provided biomolecular evidence that silk fibroin could be preserved for more than 3,000 years and could be identified in soil with only the trace of textile [20]. We subsequently reported the results of a study whose aim was to distinguish archaeological silk remains from modern silk fibres [21]. In this report, we continue this line of research and focus on identifying the invisible products of silk degradation using mass spectrometry (MS), which is a high-sensitivity, high-accuracy protein identification method. The results showed direct biomolecular evidence of silk fibroin in the soils obtained from 8,500-year-old tombs, which to our knowledge, is the first finding of its kind. Also excavated were rough weaving tools and bone needles, indicating that the detected silk may have been woven or sewn into clothing textiles.

Peptides of silk fibroin were identified in the samples from M436 and M451 by analysing the data-dependent acquisition using nano LC-MS/MS. The sample from M466 and the control samples did not contain silk peptide. Figs 2–7 present the MS/MS spectrograms of the observed peptides listed in Table 2, which all originated from the heavy chain of silk fibroin. Another important common feature of the samples from M436 and M451 is the distinctive restriction fragment, peptide GAGAGAGY at m/z 623.27837 (Theo. mass), which belongs to the crystalline area of the fibroin. The MS/MS data and the accurately deduced sequence results of the GAGAGAGY peptide are presented in Figs 2 and 6. After chymotrypsin digestion (without missed cleavages) of the silk fibroin heavy chain, this octapeptide had as many as 51 copies in one molecule (gi164448672 from NCBI). As a typical peptide with the maximum amount of copies, it was relatively easier to preserve and identify. This peptide was clearly observed in Figs 2 and 6, and was used to deduce the amino acid sequence using the b-type and y-type ion fragments. Other fibroin peptides with similar features were also detected in both samples.

Referring to our previous research [20–21], which applied similar methods to ancient samples dated from 403BC-221BC, the results showed the disappearance of the light chain. Most of the detected peptides are characteristic of the crystalline regions of the fibroin heavy chain, and few peptides from amorphous areas were detected. However, abundant information has been obtained from previous research on silk fibroin peptide fragments, including the detection of light and heavy chain fragments in fresh silk fiber [20–21]. The results indicate the decline and disappearance of detected peptide types through degradation. In this study, the data from the M436 and M451 samples shown in Table 2, indicate that most of the surviving peptides belonged to the heavy chain crystalline regions, which is consistent with our previous findings. However, there were also indications that degradation has occurred over a significantly longer time. Few remaining peptides of silk fibroin were identified in the M451 sample while fewer peptides were found in M436. The disappearance of light chain peptides, and the presence of heavy chain peptides in the crystallization regions with one in the amorphous region [17], indicated that the detected remains in the sample of M451 were those of Neolithic silk, and the M436 sample may probably contain silk protein as well.

In addition, no silk peptides were detected in the soil sample from M466, it was therefore considered as a negative sample. It is a possibility that no silk was used in this tomb. However, if silk had ever existed in M466, the result could be interpreted in several ways. The extremely low content is an important factor. The shallow-burial site could have resulted in changes in the burial environment during Jiahu’s second sub-period. These changes may have resulted in adverse preservation conditions for silk, including the introduction of extraneous matter that accelerated the degradation of the silk protein and the migration of organic remains that caused the loss or attenuation of residual silk peptides. Moreover, the negative result also eliminated the possibility that the protein came from other organisms present in the soil.

The invention of silk was significant not only to ancient China; but to all of Eurasia. As a typical early Neolithic archaeological site in China, Jiahu preserves some of the earliest evidence of human civilization. The results of this paper add silk to this list. The invisible products of the degradation of buried silk were identified in tomb soils using soil proteomics methods. The special peptides of silk protein were detected in two samples, which can be considered new and reliable evidence of the earliest silk fibre in human history. Prehistoric biomolecular evidence of silk in the early Neolithic Age discovered in the tombs of Jiahu; indicates that silk was used more than 8,500 years ago though such burial objects were not ubiquitous. If silk protein or the products of its degradation can survive in different buried environments, biomolecular data could be offered as practical and powerful evidence of the former presence of silk, reducing reliance on circumstantial evidence or guesswork based on archaeological typology. Moreover, these findings are valuable to the study of the history of silk, and advance researchers’ exploration of Neolithic Age civilization.