Increasing sustainability in palaeoproteomics by optimizing digestion times for large-scale archaeological bone analyses

# Description of data generation and experimental procedure Six bones from La Draga (Spain, Holocene, samples LD_01 to LD_06) and Baiyisha Karst Cave (China, Pleistocene, samples BKC_07 to BKC_12) were sampled for the purpose of this study. Initial sampling was divided into three sub-samples for the three digestion duration tested here (site code_sample number_3h, site code_sample number_6h and site code_sample number_18h). Samples were then processed according to the ZooMS protocol: they were demineralised in 0.6 M hydrochloric acid (HCl) for 24 hours. The HCl supernatant was then removed and samples were rinsed thrice in 100 μL ammonium bicarbonate (50 mM, NH4HCO3, hereafter AmBic) for subsequent gelatinisation in a final volume of 100 μL AmBic for one hour at 65C. Following gelatinisation, the 100 μL AmBic solution was transferred to a new microtube, to which 0.8 μg trypsin (Promega) was added for incubation at 37 °C, with mild agitation at 300 rpm (VWR, Thermal Shake lite). Digestion occurred for either 3, 6, or 18 hours. To stop trypsin digestion, 2 μL of 5% trifluoroacetic acid (TFA) was added to each sample. The digested extracts were then split in two parts for separate analyses via matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-ToF MS) and liquid-chromatography tandem mass spectrometry (LC-MS/MS). To assess any potential contamination by non-endogenous peptides, we performed extraction of laboratory blanks alongside the samples for each enzymatic digestion condition.

# 数据生成与实验流程说明
本研究采样自西班牙拉德拉加（La Draga）遗址全新世时期的6件骨骼标本（编号LD_01至LD_06），以及中国百衣沙溶洞（Baiyisha Karst Cave）更新世时期的6件骨骼标本（编号BKC_07至BKC_12）。初始采样分为三份子样本，对应本实验测试的三种酶解时长（命名格式为：遗址编号_标本编号_3h、遗址编号_标本编号_6h、遗址编号_标本编号_18h）。随后按照动物考古质谱（ZooMS）实验流程对样本进行处理：将样本置于0.6 mol/L盐酸（HCl）中脱矿24小时。弃去盐酸上清液后，用100 μL碳酸氢铵（50 mM，NH4HCO3，下文简称AmBic）冲洗样本三次，随后将样本置于终体积为100 μL的AmBic溶液中，于65℃下进行1小时的明胶变性处理。明胶变性完成后，将100 μL的AmBic溶液转移至新的微量离心管中，加入0.8 μg胰蛋白酶（Promega公司），于37℃、300 rpm温和振荡条件下孵育（仪器型号：VWR Thermal Shake lite）。酶解时长分别设置为3小时、6小时或18小时。为终止胰蛋白酶酶解反应，向每份样本中加入2 μL 5%三氟乙酸（TFA）。将酶解后的提取物分为两份，分别通过基质辅助激光解吸电离飞行时间质谱（MALDI-ToF MS）与液相色谱-串联质谱（LC-MS/MS）进行分析。为评估非内源性肽段带来的潜在污染风险，我们针对每一组酶解条件，同步设置实验室空白对照并完成提取流程。