From brains and muscles, to stomach and skin – preserved soft tissues can offer unique insights into the past, and the lives of individuals. But up to now, this treasure trove of information has been largely inaccessible to science. In the new study, the team developed the first robust method for extracting and identifying proteins from ancient soft tissues, then demonstrated its capability on archaeological human brain samples.
Alexandra Morton-Hayward, Postgraduate Researcher in the Department of Earth Sciences and lead on the study said: ‘Until now, studies on ancient proteins have been confined largely to mineralised tissues such as bones and teeth. But the internal organs – which are a far richer source of biological information – have remained a ‘black box’ because no established protocol existed for their analysis. Our method changes that.’
A key hurdle was finding an effective way to disrupt the cell membranes to liberate the proteins. After testing ten different strategies on 200-year-old human brains excavated from a Victorian workhouse cemetery, the team discovered that urea (a major component of urine) successfully broke open the cells, liberating the proteins within.
After extraction, the proteins are then separated with liquid chromatography, and identified using mass spectrometry (an analytical technique that separates proteins based on their mass and electrical charge). The team found that by coupling the liquid chromatography-mass spectrometry step with a method called high-field asymmetric-waveform ion mobility spectrometry (which separates ions based on how they move in an electric field), they could increase the number of proteins identified by up to 40%. This makes the technique a powerful approach to recover proteins from samples that are hard to analyse, including degraded or very complex mixtures.
Morton-Hayward added: ‘It all comes down to separation: by adding additional steps, you are more likely to confidently identify molecules of interest. It is a bit like dumping out a bucket of Lego: if you can start to discriminate between pieces by colour, then shape, then size, etc. the better chance you have of making something meaningful with it all.’
Using the combined method, the team identified over 1,200 ancient proteins from just 2.5 mg of sample – by far the largest and most diverse palaeoproteome ever reported from any archaeological material. The researchers point out that proteins are an ideal vehicle to navigate the recent and deep past, as they survive far longer in the archaeological record than DNA, and can tell us about the lived experience of an individual, beyond their genetic blueprint.
Working at NDM’s Centre for Medicines Discovery, the team identified a diverse array of proteins that govern healthy brain function, reflecting the molecular complexity of the human nervous system – but also identified potential biomarkers of neurological diseases, like Alzheimer’s and multiple sclerosis.
Morton-Hayward said: ‘The vast majority of human diseases – including psychiatric illness and mental health disorders - leave no marks on the bone, so they’re essentially invisible in the archaeological record. This new technique opens a window on human history we haven’t looked through before.’
Since less than 10% of human proteins are expressed in bone compared to around 75% in internal organs, this technique promises to vastly expand our understanding of ancient diet, disease, environment, and evolutionary relationships.
Professor Roman Fischer, Associate Professor and Head of Discovery Proteomics Facility in the Centre for Medicines Discovery and senior author, said: ‘By enabling the retrieval of protein biomarkers from ancient soft tissues, this workflow allows us to investigate pathology beyond the skeleton, transforming our ability to understand the health of past populations.’
The method has already attracted interest for its applicability to a wide range of archaeological materials and environments – from mummified remains to bog bodies, and from antibodies to peptide hormones.
Dr Christiana Scheib, Department of Zoology at the University of Cambridge, who was not involved with the study, said: ‘Ancient soft tissues are so rarely preserved, yet could hold such powerful information regarding evolutionary history. It is key to first develop the best way to obtain relevant information from these materials, which is what this study does. This type of fundamental experimental work is crucial for the field to move forward. The study is well-designed and I look forward to seeing what will be gleaned from the future protein data that this work has enabled.’
Find out more about the study on the PLOS One website: https://doi.org/10.1371/journal.pone.0324246