Evidence of the use of silk by bronze age civilization for sacrificial purposes in the Yangtze River basin of China
Extraction of silk fibroin from mineralized fabric or cultural relics samples
Three micrograms of mineralized sample and 5 g of ash layer sample were placed in silk protein extraction solution and dissolved in a water bath at 95 ± 2 ℃ for 45 min. After cooling, the supernatant was centrifuged, and the supernatant was extracted to conduct the iELISA.
iELISA analysis
First, 100 µL/well of the test liquid, a blank control and a negative control were added to two 96-well iELISA plates, and the plates were incubated overnight at 4 °C. The negative control was that PBS was added to the test liquid instead of silk fibroin antibody during the experiment. and silk fibroin solution was used as a positive control. Then, the liquid was removed, and 200 µL of washing buffer was added to each well to wash the unbound proteins 3 times for 2 min each. Two hundred microliters of blocking solution was then added to the wells and incubated for 2 h at 37 °C, after which 200 µL of washing buffer was added to wash the plate 3 times for 2 min each. The silk fibroin polyclonal antibody was diluted 2000 times with blocking solution, 100 µL/well of diluted silk fibroin antibody was added to test liquid and positive control wells, and the plates were incubated at 37 °C for 1 h. Then, 100 µL of washing buffer was added to wash the plate 3 times for 2 min each. The goat anti-rabbit IgG-HRP antibody was diluted 5000 times with blocking solution, 100 µL/well of goat anti-rabbit IgG-HRP antibody was then added, and the plate was incubated for 1 h at 37 ℃. Then, 200 µL of washing buffer was added to wash the plate 3 times. Finally, 100 µL/well of TMB solution was added, and the plate was incubated in the dark for 10 min. Then, 50 µL/well of 2 mol/L H2SO4 solution was added to terminate the reaction, and the absorbance of the sample was measured by a microplate reader at 450 nm.
Preparation and enrichment of simulated archeological soil sample
First, 5 mg of silk fibroin powder was weighed, dissolved in deionized water, mixed with 30 g of soil sample and placed in an oven at 100 ℃ until there was no moisture. The dried sample was removed, cooled to room temperature, and ground to ensure that the silk fibroin was evenly mixed with the soil sample. Twenty milliliters of silk fibroin extraction solution was mixed with the soil sample, and the silk fibroin was extracted at 60 °C. After 60 min, the supernatant was collected by centrifugation. To fully extract silk fibroin from the sample, the extracted soil sample was washed, filtered and centrifuged with 10 mL of silk fibroin extraction solution, and the supernatant was collected. Finally, the collected supernatant was divided into two parts: one part was selected for direct proteomic analysis, and the other part was enriched with an immunoaffinity column before proteomic analysis.
Proteomic analysis of the archaeological soil sample simulation sample
The same volume of simulated sample extraction solution I without enrichment of the immunoaffinity column and simulated sample extraction solution II enriched by the immunoaffinity column were individually added to the centrifuge tube, and an appropriate amount of 6 M guanidine hydrochloride was placed in the centrifuge tube with extraction solution, boiled in boiling water for 5 min, and centrifuged after cooling, after which the supernatant was extracted. Then, 200 µL of 8 M urea (pH 8.0) and 150 mM Tris-HCl mixture were added, the mixture was centrifuged at 12,000 × g for 15 min and then filtered, and an appropriate amount of 50 mM IAA was added. The mixture was allowed to react at room temperature for 30 min in the dark and centrifuged at 12,000 × g for 10 min. Then, 100 µL of UA buffer was added, and the mixture was centrifuged at 12,000 × g for 10 min; this process was repeated twice. Then, 100 µL of NH4HCO3 buffer was added, and the mixture was centrifuged for 10 min; this process was repeated twice. Then, 40 µL and 6 µg of trypsin were added for enzyme digestion, the mixture was shaken at 600 rpm for 1 min, and the mixture was subjected to enzyme digestion at 37 °C for 16–18 h. The liquid was transferred to a new centrifuge tube, the mixture was centrifuged at 12,000 × g for 10 min, and the filtrate was collected. The peptides were desalted using a C18 StageTip and dried under vacuum. After drying, the peptide was redissolved in 0.1% FA for LC‒MS analysis.
The peptides were separated via Thermo Scientific Easy nLC 1200 chromatography. Appropriate polypeptides were isolated from each sample by gradient elution at a controlled flow rate of 300 nL/min. The peptide was isolated and analyzed by DDA mass spectrometry with a Q Exactive HF-X mass spectrometer. The primary mass spectrometry resolution was 60,000, the parent ion scanning range was 300–1800 m/z, and the analysis time was 60 min. The secondary mass spectrum resolution was 15,000, the secondary mass spectrum of the 20 highest-intensity parent ions was triggered after each full scan, and the analysis time was 25 min.
MaxQuant 1.6.1.0 software was used to compare the raw data with 18,488 protein sequences downloaded from the UniProt protein daTablease.
SEM and X-ray 3D microscopy analysis
The longitudinal and cross-sectional morphologies of the mineralized fibers were observed by scanning electron microscopy (SEM, Sigma 300). The samples were sputtered and gilded for 60 s at an accelerating voltage of 15 kV. To assess the internal structure of the mineralized fibers, a Zeiss Xradia610Versa X-ray 3D microscope was used. A sample of the appropriate size was collected with a centrifugal tube and secured to a stand by a homemade device to ensure that it did not move when the stage is rotated. The resolution was 1.16 μm, the voltage was 80 kV, and 3DViewr software was used to extract two-dimensional microscopic high-definition images of longitudinal sections of fiber bundles.
The full experimental details and characterization of the compounds can be found in the Supplementary Information.
Materials
The silk fibroin polyclonal antibody and monoclonal antibody were prepared by our laboratory, and other reagents were purchased from commercial suppliers (Hangzhou Hua’an Biotechnology Co., Ltd., McLean Biochemical Technology Co., Ltd., Aladdin Chemical Co., Ltd., Promega Biotech Co., Ltd., Merck Chemical (Shanghai) Co., Ltd., Sigma Aldrich (Shanghai) Trading Co., Ltd., Hangzhou Fengyu Biotechnology Co., Ltd.). The mulberry silk fibroin extraction solution was a CaCl2:water: ethanol solution with a molar ratio of 1:8:2. Washing buffer (7.4 g of PBS) was prepared with 0.27 g of KH2PO4, 0.2 g of KCl, 1.42 g of Na2HPO4 and 8 g of NaCl. The antibodies and blocking solution were prepared by dissolving 2.5 g of BSA in 250 mL of 7.4 mL of PBS. The stopping solution was concentrated H2SO4 diluted to a 2 M solution. Coupled buffer (pH 8.3) was prepared with NaHCO3 and NaCl. The acid lotion (pH 4.0) was composed of 0.1 M NaAc and 0.15 M NaCl, and the alkali lotion (pH 8.0) was composed of 0.1 M Tris-HCl and 0.15 M NaCl.
