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Antonius Koller On Using Proteomics to Study Chloroplast Proteins


At A Glance

Name: Antonius Koller

Age: 36

Position: Senior staff scientist, Torrey Mesa Research Institute, Syngenta, San Diego, since 2000

Background: PhD, Biozentrum, University of Basel, Switzerland, 1990-1994. Worked on the action of immunosuppressive drugs in yeast.

Postdoc, UCSD, 1995-2000. Studied protein import into peroxisomes in yeast.

Recently published a paper in PNAS entitled “Proteomics gives insight into the regulatory function of chloroplast thioredoxins.”


How did your career lead you into proteomics?

I did my PhD at the Biozentrum at the University of Basel in Switzerland working with Michael Hall. I worked on the action of the immunosuppressive drugs rapamycin and FK506 in yeast, using mainly genetics. After that I moved to UCSD and worked with Suresh Subramani on protein import into peroxisomes, mainly in the yeast Pichia pastoris. We did genetics, basically trying to find a mutant that doesn’t import certain proteins into peroxisomes anymore. Then we cloned whatever proteins or genes we had, made antibodies, and started doing protein-protein interaction work, localizing the proteins, trying to figure out what they actually do. I got more into protein work during that time. After that, I had the opportunity to join the lab I am working in at the moment. It’s the proteomics lab at the Torrey Mesa Research Institute, which is led by John Yates.

Syngenta is going to close the Torrey Mesa Research Institute soon. Reports said that some of the staff will move on to nearby biotechnology company Diversa [Syngenta entered a $118 million research contract with Diversa in December.] Are you one of them?

We are kind of in a bubble right now. The deal with Diversa hasn’t gone through yet. There is so much uncertainty that I cannot really tell you what might happen. The only thing that’s for sure is that the institute will close.

How is the proteomics lab equipped in terms of instruments?

We have a lot of LCQ ion traps from Finnigan — those are our main instruments. We also have the usual MALDIs and Q-TOFs, a Quantum TSQ triple quadrupole from Finnigan, and a Q-TOF from Micromass.

Tell me about your research. How have you been applying proteomics to plants?

We started off doing proteomics of rice, where we analyzed proteins that we could find in rice. That was published last year [PNAS 99(18), p.11969-74, 2002, “Proteomic survey of metabolic pathways in rice”]. We used 2D gel analysis, and also MudPit analysis. We still had to develop a lot of software to handle data like that.

We also had a couple of collaborations. TMRI was sponsoring UC Berkeley, so I got into a collaboration with Bob Buchanan, which was really good. [This led to the recent publication in PNAS]. [In that study], we devised an affinity procedure to isolate thioredoxin targets that takes advantages of the mechanism by which thioredoxin reduces a specific regulatory disulfide. The mechanism requires the formation of a transient heterodisulfide bond between thioredoxin and an enzyme prior to complete reduction of the targeted disulfide. Mutation of one of the two cysteines of the thioredoxin active site — the one buried in the molecule — stabilizes the normally transient heterodisulfide, thereby covalently linking the target protein to thioredoxin via a bond that can be cleaved by DTT. We have bound mutant thioredoxins f and m [from chloroplasts] individually to affinity columns to isolate thioredoxin targets. Proteins trapped from spinach chloroplast stroma were separated on 2D gels and each spot was identified by one dimensional liquid-chromatography coupled to a Finnigan LCQ ion trap and Sequest searches. This approach enabled us to confirm the identity of more than half of the known soluble thioredoxin-regulated enzymes and to more than double the number of potential thioredoxin-interacting proteins. Many of the new targets contain conserved cysteines and are members of processes not previously known to be redox regulated.

What experiments are next?

In proteomics, most of the time we get results, but sometimes it doesn’t mean that they are actually right. The next step, at least for this project, and for a lot of other projects, is to verify the results with a different method to see if they are true.

I am actually not doing that [for this project], but it has to be done. I think it’s the same thing in drug development. You find a target, you still have to have a second method to prove whatever you just found with proteomics. I want to really stress that, because I think sometimes people believe too much [in proteomics].

Do plants pose some specific technical challenges for proteomics experiments?

I think it’s a bit harder to work with plants. For example, organelle isolations are not that easy, especially not from rice. The technology is not as developed as in mammalian cells or in yeast, and there are fewer people working in plant proteomics. For example, for immunoprecipitation experiments, there are not that many antibodies you can buy.

Also, transforming plant cells, and growing them, all these things take time. When you do an experiment with plants, and it doesn’t work, you have to wait another four weeks until your plant grows again. In yeast, if your experiment didn’t work, you are just going to do it again tomorrow.

The methods have not been totally established, but that is also due to the fact that the field is not working just on one plant. There is Arabidopsis, spinach, rice, barley, wheat, corn, etc. And a lot of methods are not readily transferable from one [species] to the other. Furthermore, to do good proteomics, one needs a good protein database. And that is probably one of the biggest drawbacks for plant proteomics. Up until now, there are only two organisms sequenced: Arabidopsis and rice. So it’s just harder to do proteomics in corn, for example.

Can you do tissue culture in plants?

Yes, you can do some culture. But I like to do experiments the natural way, meaning you don’t want to put your cells into an artificial environment. For example, when you study protein-protein interactions, you have a tag. I don’t really like tags too much, because they might interfere with your growth and your [protein] complex. Also, if you want to look at a protein complex or at the organelles in a certain tissue, you have to take the tissue and not some sort of cell culture, because you might interfere with certain variables.

Where do you see plant proteomics going, and where do you see a need for technical developments in plant proteomics?

Proteomics in general can still develop a lot of new technologies. When I talk to other people, one of the most important things is comparative proteomics, some sort of quantitation. I think that field can still improve a lot. When you look at RNA expression experiments, where you get results from 25,000 genes at once, we still have a long way to go in proteomics to do these types of experiments.

You can do [quantitation] with 2D gels, but it’s too time consuming and too labor intensive, so therefore I think that we need to have some technologies — it can be MudPIT based — that can handle the quantitation of several thousand peptides in one go.

How about protein chips?

The one technology I like in protein chips is aptamers, although I have not worked with them, I just like the idea. Proteins are so diverse, it’s going to be a problem to make a chip where you can actually have 25,000 different proteins. But somebody might prove me wrong in a few years.

What do you take from your time at TMRI?

For me it was learning how to run the mass spec, how to analyze the data. The one thing I like in proteomics, at least in our group, was that we have this diverse universe [of scientists]; there are about 15 in our proteomics group. We have a mathematician, we have a physics guy, we have analytical chemists, we have biologists, and we tried to work together to actually get to results. I think proteomics has to do that, because it’s a technology, but the technology is being used by biologists.

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