Proteomics for forensic investigation

Over the past twenty years, proteomics has not only improved in general, but has also found its application in forensic science. Although a gold standard has already been developed for DNA and RNA analyses, there is considerably more information to be obtained, write Shirin Alex, Marcel de Puit and their colleagues from the NFI in a review in Science & Justice. Alex is also the lead author of two research articles examining specific applications of proteomics in forensic science.

‘The idea behind the review article was to look at how proteomics in forensic science has developed over the past twenty years’, says Alex, a doctoral candidate at the NFI. ‘Initially it was used primarily for identifying bodily fluids, but since then many other applications have emerged.’ Proteomics has not, however, become the standard technique, largely due to already existing, well-established methods. ‘You can use proteomics to identify donors based on their biological sex, but DNA analysis is generally far more effective for this purpose. What we wanted to show is that you can obtain a great deal more information in cases where no gold standard yet exists.’ This allows you to look beyond the question of who the material came from and begin investigating precisely what happened and when.

‘The complexity of forensic science genuinely surprised me’

Hair and eye colour

Alex and her colleagues developed a proof of concept to assist in identifying mixtures of bodily fluids, for instance in cases of sexual violence. ‘With DNA analysis alone, it is difficult to determine who contributed which fluid in a mixture scenario’, Alex explains. ‘But with proteomics you can. We looked at proteins, specifically at single amino acid variants [SAAvs, ed.], which are highly specific to an individual.’ The team hypothesised that these SAAvs could be used to determine whether a fluid originated from person A or person B. ‘It is not perfect, but the proof of concept demonstrates that SAAvs can be used for source attribution.’

The team was also curious whether proteomics could be deployed to establish phenotypic characteristics such as hair and eye colour and height from blood samples. ‘That turned out to be rather too ambitious, so we opted for a simpler approach to determine an individual’s sex’, says Alex. The research generated an enormous quantity of data, so they used a machine learning model to analyse it. ‘For known or clean samples the model works very well. But when you have mock samples that have been deliberately contaminated, the model struggles.’ She concludes that it is indeed possible to elucidate phenotypic characteristics, but that DNA is far more effective. Proteomics is more valuable when applied to source attribution or establishing the timeline of events.

Discrepancy

The research sounds promising, yet the conclusions of the articles are extremely cautious. Alex explains: ‘When we wrote the grant proposal, our expectations were high, because I genuinely thought it would be much easier to apply proteomics in forensic science. The technique itself is well established and the protocols are available, so it shouldn’t be that difficult to apply them to a different research objective.’

In forensic science, however, there are many unknown factors and it is difficult to design an experiment that accounts for everything. ‘There is always a scenario you haven’t anticipated. The complexity of forensic science genuinely surprised me’, Alex admits.

There was also considerable scepticism among people in the field who use established techniques. ‘I think the discrepancy in expectations stems from the desire in forensic science to apply techniques immediately, whilst our research is still in the development phase’, says Alex. ‘The question we wanted to answer was whether there are sufficient potential applications for proteomics in forensic science.’

Alex answers in the affirmative, but a great deal more research must be done before proteomics can be used in routine investigations. ‘I think it is more suited to special cases or specific questions, such as how many donors were present and which bodily fluid came from which donor. But I am not certain that people will be willing to change their standardised routine protocols.’ The standard procedure involves removing all proteins in order to obtain as much DNA and RNA as possible. A new protocol that preserves proteins whilst simultaneously enabling extraction of DNA, RNA and proteins would therefore require considerable effort. ‘I am convinced it would ultimately be worthwhile, but I am not sure who will carry this research forward.’

Successful application

An example of the power of proteomics in forensic science was reported in 2019 in the Journal of Proteomics. ‘In the articles and in my thesis we are very cautious. But it has already been successfully applied in a rape case.’ Following a night out during which a woman had drunk too much, she left with a man in his car. The following day she reported that she had been raped after losing consciousness. The man contradicted her account, stating that it had occurred with mutual consent. She said she had passed out after vomiting in the car. Although the man had thoroughly cleaned his car, the investigators were able to find traces from the previous night. DNA was collected, but this yielded insufficient information to corroborate either account. Proteomics analysis, however, revealed the presence of partially digested proteins, providing further evidence that she had indeed vomited and corroborating her version of events. ‘Although it does not provide conclusive proof, proteomics did lend greater context to the scenario.’

On the whole, it was an enormously challenging project. Alex: ‘I did not always obtain results when following established protocols, and the project was spread across multiple institutions. But in the end it was tremendously satisfying when everything came together. Forensic science is already a small field, and forensic proteomics is an even smaller community within it. But it is a very open community and they love to brainstorm and discuss the latest research. As a first-year doctoral student it was difficult to make connections and find my place, but after a while it became much easier to build relationships.’

Alex, S. et al. (2025) Journal of Proteome Research 24(10), DOI: 10.1021/acs.jproteome.5c00098

Alex, S. et al. (2025) Science & Justice 65(6), DOI: 10.1016/j.scijus.2025.101320

Shehata, T.P., Alex, S. et al. (2025) Forensic Science International: Genetics 81, DOI: 10.1016/j.fsigen.2025.103343

Pieri, M. et al. (2019) J. Proteomics 209, DOI: 10.1016/J. JPROT.2019.103524