Using optimised sample preparation for Identification of proteins from skin tissue from a range of different Ancient Egyptian mummies

Prathiba Ravishankar, Jana Jones, Raffaella Bianucci, Do Seon Lim, Dong Hoon Shin, Mehdi Mirzaei, Paul Haynes

Research output: Contribution to conferencePoster

Abstract

Introduction: Identification of proteins from ancient tissue samples is a difficult business. Issues include questionable sample provenance and handling, and protein degradation over extended periods of time. We have compared different techniques for tissue grinding, protein extraction and protein identification, using dried modern skin as a surrogate for the ancient materials. We have applied an optimised protocol to the analysis of four skin samples from three different adult mummies from the First Intermediate Period (c. 2181-2055 BC), and four skin samples from a child mummy from the Old Kingdom (c. 2686-2181 BC). The aim of this study was to develop a sample handling protocol which maximised the amount of information we could extract from these highly valuable archaeological tissue samples.

Methods: Small samples of skin were collected, with permission, from adult and child mummies housed in the collections of the Egyptian, Museum in Turin, Italy. Modern dried skin samples were first analysed as a surrogate to use in optimising sample preparation. Proteins were extracted by either glass bead grinding or liquid nitrogen grinding, and proteins and peptides were fractionated and identified using either SDS page with in gel digestions or FASP and in solution digestions. Resulting peptides were separated by reversed-phase nano flow HPLC, then measured and fragmented using a Thermo Velos Pro or Q-Exactive mass spectrometer. Data files were searched against human protein sequences using a combination of Xtandem and Proteome Discoverer software.

Preliminary data: The analysis of ancient skin samples using proteomics approaches is different to most other experiments because the proteins are thoroughly degraded due to their advanced age, as evidenced initially by extensive smearing present when separating them on SDS – page gels. As a result, we are typically only looking at tens of proteins, rather than hundreds of proteins in a modern skin sample.
Using modern skin samples, we have found that glass bead grinding gives generally superior results to tissue grinding in liquid nitrogen, and SDS page protein separation coupled with in gel digestions gives generally superior results to FASP coupled with in solution digestion. However, there were a significant number of proteins found specifically in each of the techniques used, so a combination of complimentary techniques would most likely be the best approach if possible.
The analysis of tissues from adult ancient Egyptian mummy samples showed the identification of a large number of collagens and keratins, as expected, but also allowed for the identification of a significant number of other proteins which revealed interesting biological information. The remaining proteins identified were mostly consistent with the presence of high levels of inflammation in the tissue samples, suggesting that one of the individuals may have suffered from a chronic inflammatory condition before they died, and there is also a protein signature in a different individual strongly suggestive of the presence of pancreatic cancer.
The analysis of tissues from a child in funerary mummy basket identified a different set of proteins, which in turn revealed distinct information. A number of proteins were identified in indicative of stress response, and there was also a protein signature present strongly suggestive of a skin infection.
The information this reveals about how these people may have lived and died is very valuable in archaeological and historical terms.

Novel aspect
Optimising proteomics protocols for studying life and death in ancient Egypt.
Original languageEnglish
Publication statusPublished - 2018
EventASMS Conference on Mass Spectrometry and Allied Topics (66th : 2018) - San Diego, United States
Duration: 3 Jun 20187 Jun 2018

Conference

ConferenceASMS Conference on Mass Spectrometry and Allied Topics (66th : 2018)
CountryUnited States
CitySan Diego
Period3/06/187/06/18

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