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Q&A: Angelique Corthals on Archaeological Applications of Shotgun Proteomics

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Name: Angelique Corthals
Position: Assistant professor at CUNY John Jay College for Criminal Justice
Background: PhD, University of Oxford; Curatorial Associate, American Museum of Natural History; Lecturer in Forensic Anthropology, State University of New York at Stony Brook

Last week a team led by researchers at CUNY John Jay College for Criminal Justice and the State University of New York at Stony Brook published a paper in PLoS One on using shotgun proteomics to analyze the immune system response of a 500-year-old Inca mummy.

In the study, they used a Thermo Fisher Scientific LTQ-Orbitrap XL instrument to perform mass spec analysis on buccal swabs from two Andean mummies – a young boy and adolescent girl – identifying roughly 50 to 60 proteins including a number of differentially expressed immune response proteins indicating that the girl had been suffering a pulmonary bacterial infection at the time of death.

This week, ProteoMonitor spoke to Angelique Corthals, a forensic anthropologist at CUNY John Jay College for Criminal Justice and corresponding author on the paper, about the PLoS One study, the use of shotgun mass spectrometry in archaeological work, and its potential applications in modern day forensics.

Below is an edited version of the interview.

How common a technique is mass spec-based shotgun proteomics in archaeological work, particularly for looking at something like immune response?

It's very new. It really hasn't been done before for looking at the immune response of an archaeological specimen. I think what happens is people tend to look for the pathogen because they're interested in [whether it's] there, and if [it's] not there they infer that particular person was not suffering from the disease.

So [researchers] have been [typically] looking for DNA traces of the pathogen, but not anything else – probably because it wasn't thought possible to retrieve any [other] kind of relevant or important [molecular] information from an archaeological specimen. And to be fair, actually, originally this is what I thought. The reason I thought of shotgun proteomics … was for seeing if we could tell where the blood that we recovered from the clothing of the little boy and the lips of the maiden came from – whether it was internal blood, blood from the gut, blood from the lungs, and so on. We wanted to have information about that, and then when we looked at all of the proteins that were [identified], that's when we started doing more work on the proteins that we had seen. That's when we got the immune system response profile.

So you didn't go into this project looking to detect the immune response. You were initially just trying to determine where the blood you found came from?

Yes. We wanted to use [shotgun] proteomics, but not with the idea of the immune system response in mind. I didn't expect it to work so well.

Had you tried shotgun proteomics on archaeological samples before?

No, because I didn't have colleagues who had the facility for it. I found these colleagues [Antonius Koller, Dwight Martin, Robert Rieger, and Emily Chen] at the [Proteomics Center at the State University of New York Stony Brook], and then I was able to think about using shotgun proteomics. I knew the technology, and I had been thinking for a long time of applications for it, but I hadn't had anyone with the facility for it to work with.

In certain areas, including infectious disease, protein biomarker research has moved to trying to identify diseases by detecting the body's immune response as opposed to the pathogen or antigen itself. Did you take cues from any of that work in this project?

Yes, most definitely. I had read [about] a lot of proteomics researchers doing exactly that. And also the whole [protein] biomarker field [more generally] – I really thought that it could be done on archaeological samples.

What are the advantages of shotgun proteomics compared to the molecular techniques more commonly used in archaeological research?

The first big advantage from a technical point of view is the fact that you don't have to amplify anything. You have to do purification, but you don't have to amplify, which means that your chances of contaminating your sample are much lower – not completely gone, but much lower.

Immunoassays [which have been more frequently used in archaeological studies] are extremely susceptible to contamination. They can very easily give you a false positive. Because archaeological samples tend to be found not in the best of circumstances, so they tend to have quite a bit of contamination from the soil, from the handling, from this, that, and the other. So you have to be very aware of that. People have been doing immunoassays, and in a lot of those studies [the findings] have been found to be an artifact of contamination rather than an actual positive.

Is mass spec less sensitive, though, than, for example, PCR or immunoassays?

It could be. It makes it more tenuous in terms of what you're going to find, and that's one reason that I thought it wouldn't work as well as it did. I thought, well, we'll get what we can – and then we got a lot. I think the actual moment of truth is during the purification of the proteins. If you can do that reasonably without breaking too much the cells that have already been damaged by time, you may get a very reasonable [proteomic] profile even in samples that are not as well preserved as the one we were working on.

Are there any keys to proteomics sample prep when working with an archaeological sample?

You can try to be less aggressive in terms of purification techniques so you don't end up damaging the molecular information further than it might already be.

How difficult was it to establish a baseline, a control, for the expression of the proteins in the study?

What was really challenging was that, we had a case, with the maiden, but now we needed a control. And a control is really difficult when you have somebody who is 500 years old. So the only control we could have was the other two children. The little girl was charred by lightning, so there was no sample to be taken there, because the molecular information was burned. But the little boy was a likely candidate.

What we did was [analyze] the protein profile of the maiden and specifically the proteins of the immune system against that of the boy, who didn't show the same protein profile, and we looked to see if the difference between the two was significant. So we basically used the boy as the control when we saw that he was not suffering from the pulmonary infection. And he was not expressing the same protein profile at all despite the fact that we had very good samples from both of them.

The paper mentioned that shotgun proteomics might be similarly useful in present day forensic and criminal investigations. Is it currently being used in this way? How might it be applied?

I can't think of anybody applying it outside of toxicology – that's what it is usually used for. My personal approach to this would be that… when you have a pathogen inside a body, that doesn't automatically mean that that pathogen is what the person died of. So it would be interesting to run these same sorts of tests to determine whether the infection was latent or active at the time of death.

Now, even if the infection was active that may still not mean that was what killed the person, but that may be a better indication that this person was sick at the time of death, rather than just having the pathogen latent in them.

Is this a question that comes up commonly in forensics?

It is actually; it's a fairly common question. More common than I thought it was going to be.

Are you applying shotgun mass spec to any additional archaeological samples?

Yes. The next step is to [see if we can detect anything in] archaeological samples that are less well preserved – mainly skeletal material. We know the bone marrow is completely gone, but if we can retrieve anything from the medullary cavities of archaeological samples and see if we can detect the same kind of immune profile, that would be great.

Last year a group of researchers published a study that used mass spec-based proteomics to look at proteins in a woolly mammoth fossil (PM 12/16/2011). Do you see interest in applying shotgun proteomics growing in the field?

Very much so. I expect people are going to start thinking about it much more than before. I think most of the time when you think archaeology and you think molecular work, you think DNA, and you don't tend to think further than that. The primary responsibility of an archaeologist is really not the analysis of remains – it's really the excavation of the site, the preservation of the site. So even for people who specialize in analyzing remains, everything you read is always about DNA analysis. Now that we have this technique, I think people are going to start paying attention and start using it.

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