GoKawiilProtein extraction
Animal fossils
Powder was drilled from the dentin (Supplementary Data 1), then mixed with 1.5 ml of 0.6 M HCl for decalcification. The precipitate was washed with ultrapure water at least 3 times until the pH reached approximately 7. Then, 200 µl of 50 mM NH 4 HCO 3 was added, and the mixture was incubated at 65 °C for 3 h to extract soluble proteins. The supernatant containing the proteins was then transferred into a new centrifuge tube, and 1 µg of trypsin (Promega) was added. The mixture was incubated at 37 °C for 18 h. A 2.5% trifluoroacetic acid (TFA) solution (final concentration 0.1%) was added to stop the reaction. Subsequently, the trypsinized peptides were desalted and purified using C18 ZipTips57. For enamel samples, the powder was mixed with 1 ml of 5% HCl37 for decalcification, and the acid was replaced daily until the reaction ceased. The acid solution containing dissolved enamel peptides was concentrated using a vacuum concentrator, and the peptides were then desalted and purified using C18 ZipTips58.
Peptides were eluted into a solution of 80% acetonitrile with 0.1% TFA for MALDI-TOF mass spectrometry analysis. All sample preparations were performed in the dedicated clean room at the Molecular Paleontology Laboratory, IVPP of the Chinese Academy of Sciences in Beijing.
Hominin fossils
An acid etching method was used to extract protein from tooth enamel, modified from the process described in ref. 37. Disposable toothbrushes were used to remove surface contaminants from a small area of the enamel for etching. At the same time, the remaining teeth were wrapped with parafilm to prevent contact with any liquids. Before etching, the small enamel area was initially washed with 3% H 2 O 2 for 30 s, followed by a rinse with ultrapure water. Approximately 100 µl of 5% (v/v) HCl was placed in the cap of a 1.5-ml microcentrifuge tube. A 2-min etch was performed by immersing the etching region in the HCl solution, and the initial etch solution was discarded. A second etch, lasting 15 min, was carried out in the cap of another separate microcentrifuge tube, and the etch solution was retained. This second etch was repeated, and the etch solutions were combined. After etching, the etched area was treated with 100 µl of 50 mM ammonium bicarbonate solution for 1 min to neutralize the acid. It was then rinsed with ultrapure water for 30 s and dried. The combined etch solution was then desalted using C18 ZipTips (Thermo Fisher Scientific) and eluted into a solution of 0.1% TFA and 80% acetonitrile (ACN). The peptide mixture (50 µl) was further divided into three aliquots; one aliquot was composed of 16 µl, among which 3 μl was used for the MALDI-TOF mass spectrometry test and 13 µl was retained as backup in our laboratory; two aliquots (each composed of 17 µl) were dried for LC–MS/MS analysis in two independent laboratories. All sample preparation for the experiment was conducted in the dedicated clean room at the Molecular Paleontology Laboratory, IVPP of the Chinese Academy of Sciences in Beijing.
MALDI-TOF mass spectrometry analysis
The peptide mixture was analysed on a Bruker autoflex maX MALDI-TOF mass spectrometer. In detail, 1 µl of peptide mixture was spotted onto a MTP384 Bruker ground-steel MALDI target plate, and 1 µl of α-cyano-4-hydroxycinnamic acid matrix solution (1% in 50% ACN/0.1% TFA (v/v/v)) was added on top. They were mixed, dried, and analysed on the mass spectrometer with a m/z range of 700–3,500. Each sample was analysed in triplicate. The raw data files were processed by mMass (v5.5.0)59.
LC–MS/MS analysis
For each hominin fossil, the eluted peptides from the enamel extraction were analysed under DDA mode in triplicate, including two runs on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) at Capital Medical University, Beijing, and one run on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) at Fudan University. Both devices were coupled to an Easy nLC 1200 HPLC system (Thermo Fisher Scientific).
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