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Duncan Veal on Discovering Epicocconone, the Ethidium Bromide of the Protein World

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Duncan Veal
CEO
FLUOROtechnics

At A Glance

Name: Duncan Veal

Position: CEO of FLUOROtechnics company, centered around the fluorophore epicocconone, since 2002; Professor and associate professor of biology, Macquarie University in Sydney, Australia, since 1999.

Background: Lecturer, Macquarie University, 1988 — 1999.

Research tutor, University of Wales, 1984-1988.

PhD in microbiology, University of Reading, 1985.


At Cambridge Healthtech Institute's Beyond Genome conference in San Francisco last week, Duncan Veal gave a presentation on a molecule called epicocconone that he described as the "ethidium bromide of the protein world." The fluorophore, manufactured by Veal's company, FLUOROtechnics, is the basis for a gel-staining product called Deep Purple sold by GE Healthcare, and a protein quantification kit called FluoroProfile that is scheduled to be released on July 1 through Sigma-Aldrich. ProteoMonitor caught up with Veal after his talk to find out more about the applications of this protein-labeling molecule, and how it was discovered.

How did you get into the field of proteomics, and how did you discover epicocconone?

I fell into the proteomics field entirely by accident. My background is actually English — I trained in England as a microbiologist with the University of Reading, and I did a postdoc in the University of Wales. Then I went to Australia on a short-term contract, and when I reached Sydney I liked it so much that I never left — I've stayed there for the past 18 years. It's a great place to live. My academic interest in microbiology is actually in microbial ecology. Specifically, I've been interested in studying individuals within a population. If you think about the rest of biology, the thing that's most interesting is if you start to study populations and how those populations are constructed. It's about the diversity that you find within the populations. Microbiologists have not really got that capability to study individuals in real time within a population, and what I got into was the use of fluorescence to study those individuals within a population — using fluorescent-based techniques to study unusual individuals.

We used things like GFP, we used a lot of physiological dyes that are typically used in cell biology. We sort of imported them into microbiology and used them for flow cytometry so that we could get statistically valid numbers and sort out different populations, and also rare events within a population. We were able to, by using Fluorescence Activated Flow Cytometry, actually pull out those rare individuals.

Was there a particular problem that you were working to solve?

We were actually interested at the time of this discovery in yeast, and making better strains of yeast, and we were using flow cytometry to find new and improved types of yeast strains. One of the things we wanted to do was to make yeast cells, and part of the problem was we were working on industrial yeast strains, and industrial strains make for very low efficiency, and you can't put selected markers into yeast. So what we needed to do was to use flow cytometry to solve this problem. So basically by labeling one population red, and one population green, you could actually wait til they got together and mated, and you'd have a red-green cell, and you could go through flow cytometry and sort out the recombinants.

Well, we could find the green fluorophore from Molecular Probes, but we failed to find a source for the red fluorophore. We tried lots and lots of different sources, when one day in the lab a fungus flew in and landed on a plate and started to produce a whole suite of different pigments. It actually happened to a member of our team, a guy named Philip Bell. Because we were looking for fluorophores at the time, Phil had the presence of mind to pick up the plate and put it on the UV illuminator.

Why?

Because we're mad. He just happened to do that. We were interested in fluorescence, and we were after a red fluorophore and we had a red fungus in the lab. It doesn't sound very intellectual, but it's actually a very easy thing to pick it up and put it onto a transilluminator. If you're used to looking at fluorescence, things that are excited by fluorescence are typically blue, very rarely a green, and extremely rarely a red. This fluoresced blood red back at us, and we thought, 'This is cool. This is interesting.'

We then extracted a crude fungal extract, used it to stain the yeast cells, and the long story short is that we were able to successfully do the mating experiment, and that was published many years ago in Applied Environmental Microbiology. But we noticed when we purified the molecule, that it lost that red fluorescence, and we got that weak green fluorescence. And when we applied that weak green fluorescence and applied it to yeast cells, the yeast cells fluoresced bright red against effectively a zero fluorescence background. We thought, 'This is cool. It's great for cytometry. But what is it about the interaction between our molecule and the yeast cell that makes it fluoresce bright red?'

That's what led us to look at all of the major macromolecules within the cell, and we found that we had something that when reacted with proteins to caused a red fluorescent adduct to be produced. That's sort of the whole series of accident that led to the discovery of the molecule we named epicocconone.

Why did you name it epicocconone?

What happened was we needed the help of chemists to purify and identify the molecule, all on a shoestring budget. A guy called Peter Karuso joined the team, and Peter was able to do the structural elucidation of epicocconone. It's from a fungus that's called Epicoccum nigrum, and it's a ketone, so it's epicocconone. It was Peter who named that molecule.

We then had a molecule that was effectively non-fluorescent until it found a protein. And then another chance event — we happened to be next door to the Australian Proteomic Analysis Facility. They had some gels that they were finished with, and we bathed those gels in our fungal extract, and it lit up just the proteins in the gels, much the same way that ethidium bromide lights up DNA in a DNA gel. We thought that was quite cool. That then led us to further investigate the applications and mechanisms of the molecule for proteins. The applications are we developed it into a protein gel stain. We then licensed that to GE Healthcare who sell it as Deep Purple.

How is epicocconone different from other fluorescent labelers?

The major feature is that the molecule is not fluorescent until it conjugates with the protein. That appears to be a unique feature of it.

For example, GFP itself is a fluorescent protein. It's a big, big molecule. Our molecule is a really tiny molecule that's produced by the polyketide pathway. It's small, it's water soluble, and it's neutral. It's got a lot of nice features as a fluorescent molecule. It's not at all like GFP. GFP is more like you're using it more as a reporter of gene function. Our molecule is really specifically to detect, track, and to quantify proteins, because that reaction is quantifiable by fluorescence output.

The other key feature is the reversibility of the staining. That's really important for proteomics, because you don't just want to quantify the protein and detect the protein. You want to do other things with the protein like mass spec in order to identify it, or Edmund sequencing, or antibody staining. One of the nice things about epicocconone is it can both go onto the protein, but it will also come off, allowing you to do all the downstream processing.

How do you get it to come off?

For gels, you want the label to go on, and you want it to stay on. You do that by lowering the pH to pH 2.5, and then effectively it doesn't come on and off. If you raise the pH, it effectively shifts the balance to the equilibrium of it coming on and off.

What are your next steps after this?

Well, we've got Deep Purple out as a product for gel staining, and also for blot staining as well. That's the GE Healthcare product. We've got a product being released called FluoroProfile. That's coming out July 1st through Sigma. Because we've got an entirely new method of quanitifying proteins, that particular method of quantifying proteins isn't subject to the same sort of interfering compounds that the classical methods like Lowry or Bradford protein assays are. For example the Lowry depends on the reduction of copper. So you have a reductant there that interferes with the quantification. The Bradford depends on detergent interactions, and the detergent interferes with downstream reactions. But we've happened to have stumbled across something that works with an entirely different method that doesn't interfere to nearly the same extent as reductants and detergents. That's particularly important in things like 2D gel proteomics where you have things like DTT, high levels of detergents in your sample buffer.

We're actually also making epicocconone available for other people. We see is as being a packed-full technology, and we want to enable other people to develop technologies around epicocconone.

For the quantitative kit, how does it work?

You run it against a standard curve, and you can run it on a microtiter plate or in cuvettes.

Is the kit comparable at all to the ICAT or iTRAQ labeling kits?

It's an interesting question. I'm not sufficiently a technical expert enough to comment on whether they're comparable. People have said to me that they think it will work in the same way as the other labeling kits. What we're saying is, 'Here's the molecule. Go for it.'

What's the cost of the molecule?

You can go to GE Healthcare or Sigma to find out cost of the kits. The cost of the molecule is $750 per milligram. It's very powerful stuff. You only use tiny amounts for quantification.

It's an unusual molecule because of its spectral characteristics, and I think it will have lots of applications as a classic protein molecule because it's small, water soluble, and it's got a large Stoke's shift. It's quite unusual in Australia to spin off companies, and it's really nice that we've had a supportive university that's enabled us to spin off the company.

This molecule and this technology I just see as having such broad applications, and we really want other people to become aware of it and to start thinking of ways in which they can start using it.

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