Ancient Egyptian Mummy of the first intermediate period

I joined in the international collaboration on the Ancient Egyptian Mummies (Please see below).

The news was reported by "Seeker"
as "Skin Proteins Reveal How Mummies Died"


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Published as peer reviewed paper ("Identification of proteins from 4200-year-old skin and muscle tissue biopsies from ancient Egyptian mummies of the first intermediate period shows evidence of acute inflammation and severe immune response") in


Abstract:
We performed proteomics analysis on four skin and one muscle tissue samples taken from three ancient Egyptian mummies of the first intermediate period, approximately 4200 years old. The mummies were first dated by radiocarbon dating of the accompany-\break ing textiles, and morphologically examined by scanning electron microscopy of additional skin samples. Proteins were extracted, separated on SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels, and in-gel digested with trypsin. The resulting peptides were analysed using nanoflow high-performance liquid chromatography–mass spectrometry. We identified a total of 230 unique proteins from the five samples, which consisted of 132 unique protein identifications. We found a large number of collagens, which was confirmed by our microscopy data, and is in agreement with previous studies showing that collagens are very long-lived. As expected, we also found a large number of keratins. We identified numerous proteins that provide evidence of activation of the innate immunity system in two of the mummies, one of which also contained proteins indicating severe tissue inflammation, possibly indicative of an infection that we can speculate may have been related to the cause of death.

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Also presented in 64th ASMS Conference on Mass Spectrometry and Allied Topics (June 5-9, 2016, San Antonio, Texas)

Prathiba Ravishankar, Dylan Xavier, Fallen Teoh, David Handler, Mads Foged, Mehdi Mirzaei, Dong Hoon Shin, Rafaella Bianucci, Jana Jones, Paul A. Haynes

Title
Proteomics and Ancient History - Identification of proteins from skin and muscle tissue from Ancient Egyptian mummies

Introduction
Identification of proteins from ancient tissue samples is challenging and problematic due to degradation over time, and issues of sample provenance. We aimed to perform a proof-of-concept study on a series of eight skin and muscle tissue samples collected from ancient Egyptian mummies, mainly of the First Intermediate Period (c. 2181-2055 BCE). The scarcity of the samples, and the logistic difficulties in acquiring them, meant that there was not sufficient material available to optimise sample preparation and perform trial experiments.  Our goal was to show that we could identify proteins from material this old, and in this condition. Any information this reveals about how these people may have lived and died is very valuable in archaeological and historical terms.

Methods
Small samples of skin and muscle were collected, with permission, from mummies housed in the collections of the Egyptian, Museum in Turin, Italy. Samples were rehydrated in SDS Page sample buffer, and homogenised using high-speed agitation. Extracted proteins were visualised using coomassie blue staining. Triplicate gel lanes containing protein were exercised into 16 fractions, and in gel digested with trypsin. Resulting peptides were separated by reversed-phase nano flow HPLC, then measured and fragmented using a Thermo Q Exactive mass spectrometer. Data files were searched against human protein sequences using a combination of Xtandem and Proteome Discoverer software.

Preliminary data
In contrast to most shot gun proteomics experiments, the issue in this work is not managing mountains of data. The proteins in the tissue samples are thoroughly degraded, as evidenced by the extensive smearing present when visualising SDS page gels. This is confirmed by the low number of peptides and proteins identified in the samples. We are typically looking at less than 50 proteins reproducibly identified, whereas a similar analysis of more recent human skin samples reproducibly identifies hundreds of proteins.
Preliminary analysis of the first few samples has shown that we can easily identify numerous keratins and a large number of collagens, as expected. This ties in well with microscopic analysis of similar samples obtained from the same mummies, which clearly showed the presence of collagen fibres still intact. The perhaps more interesting feature of our data so far is that we have also identified five proteins which are all consistent with the presence of inflammation in the tissues. This suggests that this person may have suffered from chronic inflammatory condition before they died. This is exactly the type of clue which we were hoping to find when we initiated this study.
We will present details of all the proteomics analyses we have performed on this set of tissue samples. Analysing a larger number of samples will allow us to focus on what is common between them, and also what stands out as being unique to individual samples. We will also discuss this information in the context of other analyses which are ongoing on samples from the same mummies, which includes histological and microscopic examinations.

Novel aspect
Using proteomics to uncover clues about life and death in ancient Egypt.


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