Tryps-IN: A streamlined palaeoproteomics workflow enables ZooMS analysis of 10,000-year-old petrous bones from Jordan rift-valley

Poor preservation of collagen in dry and/or arid environments has hindered the application of Zooarchaeology by mass spectrometry (ZooMS) analysis in many regions of the world, and as a result many zooarchaeological investigations have relied exclusively on the morphological assessment of fragmentary remains, due to the inadequate preservation of biomolecules. The climatic conditions of Southwest Asia include extreme temperature fluctuations unconducive to preservation of proteins and DNA. We performed zooarchaeological analysis of remains from the 10,000-year-old site of Shkārat Msaied in Jordan and sub-sampled twenty-eight petrous bones, the hardest bone in the mammalian skeleton, for species identification by ZooMS. Using an unconventional and simplified extraction protocol we call Tryps-IN, in which digestion was performed without removal of the demineralising EDTA, we taxonomically identified several fragments, outperforming the established ZooMS work-flow. A subset of identifications was subsequently confirmed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) protein sequencing. The new methodology presented here opens the possibility of further bioarchaeological investigation of other fragmentary faunal assemblages within this region of archaeological significance.干旱乃至半干旱环境中胶原蛋白保存状况不佳，阻碍了基于质谱的动物考古学（ZooMS）分析在全球诸多区域的应用；受生物分子保存不足的限制，此前多数动物考古研究仅能依赖破碎遗存的形态学鉴定。西南亚地区的气候存在极端温度波动，不利于蛋白质与DNA的保存。本研究对约旦什卡拉特姆赛义德（Shkārat Msaied）遗址——一处距今10000年的考古遗址——出土的遗存开展动物考古学分析，并采集了28块哺乳动物骨骼中最坚硬的岩骨，用于基于ZooMS的物种鉴定。我们采用一种名为Tryps-IN的非常规简化提取流程：该流程无需移除脱矿用乙二胺四乙酸（EDTA）即可完成酶解，成功对多件遗存碎片完成分类学鉴定，其效果优于现行标准ZooMS工作流程。随后，我们通过液相色谱-串联质谱（LC-MS/MS）蛋白质测序技术，对部分鉴定结果进行了验证。本文提出的新型方法，为该具有考古学价值的区域内其他破碎动物遗存组合的进一步生物考古研究开辟了可行路径。