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. As a result, many zooarchaeological investigations have relied exclusively on 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 Shka over bar rat 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 an established ZooMS workflow. 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.