GoKawiilArchaeological site and samples
Bianfu Cave (26° 27′ 43.65′′ N, 100° 09′ 55.45′′ E) is in Heqing County, western Yunnan Province in southwestern China, approximately 2 km south of the Yinhe River, a secondary tributary of the Yangtze River (Fig. 1a). In 2019, the Yunnan Institute of Cultural Relics and Archaeology and other cooperative institutes conducted excavations on this site. The stratigraphy comprises Holocene deposit, artefact-bearing cultural deposit and underlying cave-fissure deposit. The cultural deposit is further divided into upper (layers 3–6) and lower (layers 7–12) cultural layers (Fig. 1b). A Bayesian age model combining optically stimulated luminescence and U-series dates yielded a time range spanning approximately 190 ka to 70 ka1, making it the earliest Palaeolithic site discovered in western Yunnan. All cultural layers yielded lithic artefacts, totalling 1,366 stone artefacts, including both core and flake tools1. More than 64,000 mammal fossils were recovered from the cultural layers, among which more than 60,000 were fragmentary bones. On the basis of the limited number of morphologically identifiable mammal fossils, more than 100 individuals of deer and bovids were discovered, together with small numbers of rhinoceros, hyenas and other animals1.
Four hominin teeth were unearthed in the middle of layer 7 (between 148–139 ka and 142–134 ka)1, suggesting a high likelihood of finding more hominin fossils in this site. The four hominin teeth included a lower left lateral incisor (I 2 ), a lower left fourth premolar (P 4 ) and two lower right second molars (M 2 ). Morphological analysis showed that the lower M 2 teeth exhibited close morphological affinity with those of Xiahe and Laos Denisovans, as well as the East Asian late Middle Pleistocene archaic Homo from Hualongdong1. Two of these teeth, a lower fourth premolar (YHB3518) and a lower second molar (YHB3075) (Fig. 1c,d), were selected for proteomic analysis to further confirm their taxonomic attribution. An acid etch approach was used for analysis of the enamel, whereas dentine powder was drilled for protein extraction, resulting in one enamel fraction and three dentine fractions for liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) analysis.
After manual inspection and traditional morphological measurement of more than 60,000 bone fragments on the basis of bone size, cortical thickness, articular surface morphology, muscle attachment marks on long bone surfaces, vascular impressions on cranial internal surfaces and so on, 22 fragments were considered as potential hominin remains, five from the upper layers and 17 from the lower layers. Before conducting protein extraction on these 22 remains, we randomly selected 36 bone fragments from the upper layers and 18 from the lower layers for preliminary ZooMS analysis to assess protein preservation of the site. Thus, a total of 76 bone fragments were analysed by ZooMS in 6 batches, with each batch including an extraction blank and a positive control (either modern camel bone or dentine powder) (Supplementary Data 1). After ZooMS identification, 3 hominin remains and 14 mammalian samples from different layers were submitted to LC–MS/MS analysis, including the corresponding extraction blanks. To obtain a more comprehensive proteome of the hominin remains at this site, we performed further proteomic extraction on the parietal BFD767, yielding another three fractions for LC–MS/MS analysis. We also performed U-series dating to estimate the minimum age of three hominin bone samples.
U-series dating
U-series dating was conducted on three hominin bone samples and their attached carbonates. For each sample, a series of 5–11 spot pairs were analysed using a laser ablation system coupled to a multicollector inductively coupled plasma mass spectrometer (LA–MC-ICPMS). The LA system (RESOlution-LR, Applied Spectra) was equipped with a Coherent COMPex Pro102 ArF Excimer laser source and an S155 large-format sample pool. The Neptune double-focusing MC-ICPMS (Thermo Fisher) had nine Faraday cups and a secondary electron multiplier, allowing simultaneous measurement of several isotopes over a relative mass range of 17%. The Neptune interface was upgraded with a Jet-sample cone, an X-skimmer cone and a high-efficiency dry pump to improve the ion transport efficiency. By tuning the analytical parameters (carrier gas flow, torch position and zoom optics) of our LA–MC-ICPMS, we could routinely obtain 238U signals of approximately 0.8–1.0 V and 232Th/238U ratios of approximately 90–95% on the international standard NIST 612, with a spot size of 173 μm, a pulse repetition of 10 Hz, an energy fluence of 5 J cm−2 and a scan speed of 3 μm s−1. We conducted a preablation step to remove impurities from the sample surface. Then, each spot was ablated for 120 s at 10 Hz pulse repetition and 5 J cm−2 fluence. With signal intensities of 238U and 230Th, the spot size ranged from 50 μm to 180 μm. These laser ablations produced a crater approximately 150 μm deep. We measured isotopes 230Th and 234U using a secondary electron multiplier and simultaneously measured 232Th, 235U and 238U in Faraday cups47,48,49,50. For each spot, around 150 cycles were performed to acquire isotopic data, with the first 40–50 cycles collected before sample ablation and used as the gas blank. An in-house fossil bone standard (RM-B1) and a carbonate standard (RM-C1) were used as the matrix-matched standards for calibration of laser-induced elemental and isotopic fractionation for the hominin bones and carbonates, respectively.
ZooMS screening
Around 100–200 mg of the sample was used for protein extraction with the conventional acid-insoluble approach51. After demineralization in 0.6 M HCl, the pellet was incubated with 50 mM ammonium bicarbonate (ABC) at 65 °C for 3 h. The supernatant was then collected and digested with trypsin. The digested peptide mixture was desalted using Pierce C18 Pipette Tips (Thermo Scientific), and then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis.
Palaeoproteomic extraction
For the identified hominin parietal BFD767, a second round of protein extraction was conducted to obtain more proteins. The protocol was modified from that reported in a previous publication52. Another subsample (around 300 mg) was demineralized in 0.6 M HCl, and the acid supernatant was collected and ultrafiltered through an Amicon Ultra-4 (3 kDa) centrifugal filter unit. After the filtration unit had been washed with buffer (50 mM ABC), we obtained the ‘acid-soluble fraction 3KD’ by dissolving the proteins retained on the filter in 50 mM ABC. The pellet was further incubated in 50 mM ABC at 65 °C for 3 h. The supernatant was reduced with Tris(2-carboxyethyl) phosphine HCl (final concentration 0.1 M) at 56 °C for 30 min and alkylated using iodoacetamide (final concentration 0.1 M) at room temperature in the dark for 30 min. The supernatant was divided into two aliquots, one for trypsin digestion and the other for elastase digestion. The trypsin aliquot was ultrafiltered through an Amicon Ultra-4 (3 kDa) centrifugal filter unit, washed with buffer (ABC) and dissolved in 50 mM ABC; this was referred to as the ‘acid-insoluble fraction trypsin’. The elastase fraction was ultrafiltered through an Amicon Ultra-4 (3 kDa) centrifugal filter unit, washed with buffer (Tris-HCl solution) and dissolved in 50 mM Tris-HCl; this fraction was referred to as the ‘acid-insoluble fraction elastase’.
... continue reading