Professor of protein mass spectrometry
University of Southern Denmark
At A Glance
Name: Ole Jensen
Position: Professor of protein mass spectrometry, University of Southern Denmark, since 2004. Associate, assistant professor, since 1997.
Background: Postdoc, Matthias Mann's laboratory, European Molecular Biology Laboratory, 1994-1997.
PhD in biochemistry and biophysics, Oregon State University, 1994.
The Danish Proteomics Society plans to hold its first meeting on Dec. 8. ProteoMonitor decided to talk to one of the leaders of the society to find out about his work and his vision for the organization.
What is your research background, and when did you get into proteomics?
I started here in the University of Southern Denmark as an undergraduate. I graduated in 1990, and already then I worked with protein chemistry and mass spectrometry using plasma desorption mass spectrometry. That was the time when MALDI and electrospray ionization came out. So during my PhD studies at Oregon State University in the US, I used those techniques to study proteins.
For my PhD, I was mainly looking at protein-nucleic acid interactions, and protein chemistry. We wanted to study the mechanism behind the recognition process for nucleic acid-protein binding. Certain proteins recognize certain sequences in DNA, and we were interested in a DNA repair enzyme that recognized damaged DNA. Basically, we developed a method for cross-linking the nucleic acid to the protein, and then we used mass spectrometry to map out where the cross-linking had taken place, and tried to find the active site of the protein.
I finished my PhD thesis in 1994, and that's when I moved to the EMBL in Heidelberg, Germany, and joined Matthias Mann's group. That's when I got into proteomics for good. That was around the birth time of proteomics, and we were doing a lot of work with protein identification from gels using MALDI and electrospray ionization. We did some of the pioneering work in sample preparation for identifying proteins from 2D gels, for example.
Quantitation was done based on the 2D gel images. And then our goal was to develop sensitive, mass spectrometry-based methods for identifying the protein spots from gels. So I worked mainly with MALDI peptide mass mapping at the time, developing automated and robust methods for analyzing many proteins.
We did some yeast proteome work — we had a large-scale study of yeast — at that time we identified, I think, 150 proteins on a 2D gel. It was a different time. At that time, identification of 150 proteins was a big event.
Were you cataloguing proteins, or were you also looking at disease proteomics?
These first experiments were what you call cataloguing, because it was all about developing the methods. Before 1995, we didn't have any methods where we could look at proteins systematically from gels. So the first challenge was basically to identify proteins, and to try to develop robust and fast tools for doing that.
Then the next level came in the late '90s when we looked at protein complexes. For example, we looked at the post-synaptic density in brain tissues, and the spindle pole body in yeast, to try to resolve some molecular complexes, just for understanding molecular cell biology events.
After I left Matthias Mann's lab, I returned to Denmark. Then I started up my own research here, which focused on establishing some of the methods I had established at EMBL for protein identification, and then moving into phosphorylation site mapping. Since then I have been focusing on post-translational modifications, developing affinity enrichment methods and quantitative methods to study phosphorylation and other modifications.
What got you interested in phosphorylation mapping?
Well, it was already evident at that time that post-translational modifications are very important for protein function, since they modulate protein interactions, and they determine the activity of proteins, and they also are sometimes involved in localizing proteins to the right compartments inside of cells. So from a biological point of view, phosphoproteins and cell signaling were very important at that time, but there was really no proteomic effort in that direction yet. It was only about to start. By that time we knew how to identify proteins, and we could also map big complexes of proteins, so the next challenge was to try to look at the modifications.
So some groups went on and started working more on the quantitation aspects in proteomics, and some other groups went off and did phosphorylation mapping and mapping of other modifications. And since we have so many modifications around in proteins, there's enough to do for everybody. But phosphorylation was sort of top on the list, which is still the case.
What are you working on presently?
We study protein phosphorylation in various organisms. We have worked a lot with plants — Aribidopsis — where we have mapped out plasma membrane phosphoproteins using a strategy which we call 'shave and conquer,' which we published in 2003.
Basically, the idea is to first isolate the organelle — in this case the plasma membrane — and then in order to get access to the phosphorylated domains of membrane proteins, we need to do a trick. When you prepare a plasma membrane sample, you form vesicles. Usually the plasma membrane is oriented in a way such that the external part is oriented out, so we can not have access to the phosphorylated domains because they're hidden inside the vesicles. So the trick was introduced by a postdoc who found out that if you treat with a certain detergent, you can flip the vesicles inside-out. Then trypsin has access to the phosphorylated domains, so we can shave the inside-out vesicle that way.
And then the next step is to use the IMAC — immobilized metal affinity chromatography — which we already had worked on for a number of years. We then use the IMAC technique to affinity purify phosphopeptides. So in this case, we could do a large-scale study of phosphorylation sites in plasma membrane proteins. So we found several hundred phosphorylation sites.
What were some of the more interesting proteins that you found in Arabidopsis?
We could actually find a whole set of receptor-like kinases. I think about 25 percent of all the membrane proteins we found were part of this receptor-like kinase family. That was very interesting because very few phosphorylation sites had been mapped in that family, and we could then contribute a whole new set of phosphorylation sites. And it turned out that many of the phosphorylation sites are actually between domains in the proteins.
So the proteins are organized in domains, and many people think that phosphorylation usually takes place inside the domains, but we found that they are between the domains, which is an interesting observation.
We think that [the phosphorylated sites] are involved in the recruitment of adapter molecules, and involved in signaling and scaffolding of the signaling apparatus. So it's sort of a barcode — a phosphorylation barcode — for recruiting cofactors and adaptor molecules that can then mediate the signaling event. So we think that they're important for recognition of interaction partners.
Are you working on other organisms besides Arabidopsis?
Yes. We have in the meantime refined the technique further. This year we published a nice paper on yeast. We have done a functional phosphoproteomic analysis of yeast after hormone treatment. This was done in a quantitative manner so that we could not only determine phosphorylation sites, but we could also quantify changes in phosphorylation in 500 proteins. This way we could identify many of the signaling proteins involved in the hormone signaling inside the cell, in an unbiased manner.
What projects are you working on for the future?
We have many projects. One of them is to apply this technology to study human phosphoproteins, but we're also continuing with yeast because it's a very nice model for studying phosphorylation and signaling, because so much is known, and it's very easy to grow.
But the main aim of the group is to develop new analytical technology. We focus mainly on sample preparation methods to get very selective and specific enrichment of modified proteins and peptides prior to analysis by mass spectrometry. We are trying to integrate sample preparation methods, mass spectrometry methods, and bioinformatics methods to do large-scale analysis of protein modifications.
What kind of cells do you think you'll analyze in humans?
The big challenge is, of course, to be able to analyze tissues, and also disease tissues. But that's for the future. That's what we're eventually aiming for. But we would like to develop more sensitive methods, because right now the combination of methods we have will have to be further refined to be able to study primary tissues and to get a deep phosphoproteomic analysis performed.
How are you involved with the Danish Proteomics Society?
I'm actually trying to set up the society. It's not really formalized yet. We're going to have the first meeting next week, on Dec. 8. We'll have a symposium.
What is your vision for the organization?
The basic idea is to have a network of people here in Denmark to then be able to have a more or less formalized forum for exchanging information. Since Denmark is a rather small country, we would like to link up to some of the other Scandanavian countries like Norway and Sweden and Finland.
Sweden has a proteomic society, and Norway and Finland are working on setting up theirs. So one aim is to try to have a Scandanavian network of proteomic researchers, and then, of course, to also have a network with the rest of Europe through the European Proteomic Association. I'm also involved with that, representing Denmark for now.
Also, I've been elected a member of the HUPO council, beginning in 2006.
So I'm trying to coordinate things. We'll start with this symposium on Thursday of next week, and then see how much interest there is. Already more than 50 researchers have signed up for the meeting.
I think the main emphasis right now for EuPA is to establish the infrastructure for doing collaborative work. That would rely on being able to exchange data between labs. This comes back then to standards for data exchange. There's a lot of effort right now coordinated by the European Bioinformatics Institute and HUPO, and by EuPA. That's something everybody's working for right now. I think once that is in place, you can begin to think about collaborative projects.
As always, funding is an issue. For big collaborative projects within the European Union, we would apply for funding from the European community. And then also we will identify some sources of funding for the exchange of students, because there's a huge need for education in the field of proteomics, not only for training people to work with mass spectrometry and separation methods, but also for interpreting the data that comes out of large-scale experiments. There is a big need for bioinformatics, especially proteome bioinformatics, to interpret mass spectra, to annotate modifications in proteins, and to compare protein sequences and basically to be able to handle and compare large data sets.
Is there quite a lot of proteomic work going on in Denmark?
Yes, it's pretty well established now. We have groups at six universities now, out of a total of 12 universities. There are six universities that have serious programs in proteomics. Some of them are focusing more on plants, and some are focusing more on microbes and yeast for biotechnology applications.
There's a lot of research on husbandry using proteomics to look at meat, for example. Bacon is a big export article, so there's a lot of interest in applying functional proteomic methods to quality test meat, and also to optimize conditions for breeding pigs and cows. They're trying to understand why piglets get diseases, for example, and how to improve the survival rate for piglets. They're also trying to improve the tenderization of meats. You can use proteomics in all these areas.
They're also investigating plants and plant pathogen interactions. There are many, many interesting projects around and since the University of Southern Denmark is sort of the main center for protein mass spectrometry and proteomics, we get in touch with many of these other proteomic projects as well, because we can help them out with advanced mass spectrometry analysis. So it's very exciting, actually, to be in a small country because we get in touch with many researchers here and we have many good collaborative projects.
In large-scale proteomics, I think it's important that many people work together. You have cell biology, you may have some medical aspects involved, you have the protein analysis and mass spectrometry involved, and the bioinformatics in the end. All this has to come together in an integrated way. This is why research groups have to work together to get the best outcome for large-scale projects.