Bioarchaeological proteomics: identification of proteins from skin and muscle tissue from Ancient Egyptian mummies shows evidence of acute inflammation and immune response

Proteins are more resilient molecules than DNA, which means there are certain advantages in analysing ancient proteins rather than ancient DNA. Shotgun proteomics analysis involves the characterisation of the protein profile of a given biological sample using nano flow liquid chromatography – tandem mass spectrometry, which generates large scale data from miniscule amounts of material. Archaeological research is often limited to tiny amounts of very precious samples available for scientific analysis. However, there is no validated proteomics workflow in the published literature that could be applied to ancient human skin tissue samples.

We present here details of our work on the optimization and application of sample preparation methods for shotgun proteomics profiling analysis of ancient skin samples, by first using modern human skin tissue as a surrogate. We have tested two different grinding techniques, glass bead grinding and liquid nitrogen grinding, for use in the process of protein extraction. We have also tested two different protein digestion techniques, SDS-PAGE and in-gel digestion, and filter-aided sample preparation (FASP) with in-solution digestion.

Using an optimized approach, we have subsequently performed a shotgun proteomics analysis to investigate the protein profile of a set of four ancient human skin samples. These were sampled from an ancient Egyptian child in funerary basket of the Old Kingdom (2200 – 2000 BC). The results of these analyses are quite distinct from those of our previous work on skin samples from ancient Egyptian mummies. In addition to the immune response and inflammation related protein is, we identified numerous proteins related to skin infections. Thus, we have been able to generate unique archaeological information using molecular analysis techniques, including an indication of the health state of the individual at the time of death.