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Matthias Wilm, EMBL, on Studying Protein Complexes in Eukaryotes

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At A Glance:

Name: Matthias Wilm

Age: 40

Position: Group Leader, Bioanalytical Research Group, EMBL, Heidelberg, Germany, since 1998

Prior Experience: Diploma (equals BS/MS) in physics, University of M nster, Germany, 1989

MD, University of M nster, Germany, 1991

PhD, EMBL, Heidelberg, and University of M nster, 1996. Developed a nanoelectrospray ion source in Matthias Mann’s group.

Postdoc, EMBL, 1996-1998

Recently published a paper in Nature Biotechnology entitled “An efficient protein complex purification method for functional proteomics in higher eukaryotes.”

 

How did you get into proteomics?

I studied physics at the University of M nster in Germany and also completed a medical degree. For my diploma thesis [in physics] I joined the lab of Professor [Alfred] Benninghoven in 1988. He was using mass spectrometers to do surface analysis, just around the time when the two soft ionization techniques that revolutionized biology five or ten years later were discovered. While I was working there, one of them [MALDI] was discovered in the medical physics department in M nster — I was in the physics department — by [Franz] Hillenkamp [together with Michael] Karas.

At that time I worked on the electrospray mechanism. My project was to use electrospray to cover surfaces with a sample in a very homogeneous way, like spraying paint on a car — which also uses electrospray, but in an uncontrolled way. The discovery that electrospray could be interfaced to a mass spectrometer to generate large ions was made in 1988 by John Fenn in the United States. It was quite a good coincidence that I was working on the same thing. I described the mechanism of the electrospray process in a theoretical way, and could demonstrate that an electrospray ion source could be made much more sensitive.

After my diploma work I had to finish my medical degree, which brought me to Paris and Dortmund [Germany] for a year. Professor Benninghoven then put me in touch with Matthias Mann, who at that time was about to start his group at EMBL, using mass spectrometry for biological research. So we started half a year later, in 1991, in Heidelberg, he as a group leader and I as his graduate student. In his lab, I could implement my electrospray theory on a mass spectrometer, and that led to the creation of the nanoelectrospray source, which at that time was about 100 times more sensitive than any other electrospray source on the planet. Then we started to analyze intact proteins and tried to sequence peptides by mass spectrometry.

But there was still one problem to solve, and that was reading the sequences from the fragment spectra, because that was a very different process from classical chemical sequencing, where you read one amino acid after the other in a chemical degradation cycle. Matthias Mann came up with what is now called the sequence tag algorithm, and then we could identify peptides, and with the peptides the proteins. Those were the two things we needed to get what is today called proteomics going: a highly sensitive [analytical] method and a method to identify sequences in the databases.

After my PhD, I stayed for another two years as a postdoc. Then Matthias Mann left to take up a professorship in Odense in Denmark. EMBL needed a successor for the mass spectrometry group, and they offered me the position in 1998.

What does your group focus on?

My group is called Bioanalytical Research Group. EMBL has now two mass spectrometry groups: One is a clean-cut proteomics group, the Proteomics Visitor Facility, that uses 2D gels to separate proteins and display their quantities and mass spectrometry to identify them in high throughput, whereas our group is a bit more on the method development side. Proteomics activities have been driven mostly by mass spectrometry in the past, but currently they are driven by the biochemical techniques. They cover two sides: One is precise quantitation of proteins. We are active there but less active than in the second field, which EMBL has always been strong in, and that is functional proteomics, the systematic activity to try to put proteins into their functional context. The key technique here is analyzing protein complexes. If two proteins function within one biochemical pathway, they often evolved to have an affinity to each other. If you want to find proteins which have a functional relationship, you must be able to purify these non-covalent protein complexes in a very good way. That is what molecular biologists have always done, but two inventions coming from EMBL have improved the technique to purify protein complexes in the last two or three years. One is tandem affinity purification, or the TAP method.

My group didn’t invent TAP, that was the group of Bertrand Séraphin, but when he came to our lab showing the first complexes on a gel, I said ‘that is extremely important, we are going to pursue this project as the main project within our lab,’ and in the following year supported it with all our capacity.

Wasn’t this technique also used by Cellzome in their 2002 Nature paper?

That’s correct. Cellzome resulted from this after we pursued this project for more than a year and the investment bankers thought that this was a very good project to put money into. I am one of the founders, and so is Bertrand.

Recently we were successful in porting the TAP method to higher eukaryotes. It was originally developed in yeast, and in the following years we supported groups within EMBL that tried to use the TAP technique, but it never worked as well in higher eukaryotes as it did in yeast. The reason was probably that in yeast they could eliminate the native protein, having the tagged protein as the only protein of this kind in the cell, whereas in higher eukaryotes, they couldn’t eliminate the native protein. What we call the iTAP technique is a combination of the original TAP approach plus RNAi, interfering RNA, to knock out the native protein. We have a very good website with protocols [and the method was published in Nature Biotechnology in January].

Licensing is handled by EMBLEM, which is the technology transfer arm of EMBL. I heard that they got a very good offer, but I don’t know what decision they will make.

What do you use iTAP for?

The projects always depend on the interest of the groups we collaborate with. It will be used in investigating nuclear transport: there are several development projects coming where people study chromatin organization in the nucleus, and one project in mouse that I cannot specify yet.

What else do you work on?

We also study posttranslational modifications of proteins because mass spectrometers are the best tools to do this. That’s a technical reason. The second reason is, posttranslational modifications — not only phosphorylation, but acetylation as well — are switches for function on-function off.

How are you equipped in your group, and how many people do you have?

We have four [mass spec] machines: an API III triple quadrupole machine from ABSciex, an old MALDI and a very new MALDI machine, both from Bruker, and a Q-TOF electrospray machine from Micromass. We have only two people working on the mass spectrometers, a postdoc and a predoc, but we will get a new postdoc soon. We also have one predoc biologist working with the group of Elisa Izaurralde to get some biological projects going.

What is the Proteomics Visitor Facility?

At the origin was a [decision] by the director general of EMBL to do large-scale proteomics at EMBL. That was at a time when we did not have new enough instruments to support this technically. I had some friends at Micromass who heard about this. This must have triggered a discussion within Micromass whether it wouldn’t be a feasible to sponsor a visitor activity at EMBL — we also wanted to allow European scientists to come to EMBL and see the technology and work with it a little bit. Micromass was the main contract partner of EMBL and brought BioRad with them. They donated close to the entire equipment of this lab, which has four staff members. It has a double function: it is a core facility for EMBL, so EMBL scientists can run larger projects in the facility. Visitors are restricted to one or two weeks at a time, because the lab has a limited capacity. In the last two years or so, we have had about 300 visitors. Many just passed by and looked at the instrumentation only for one day to see what it can do. A minority stayed for a week or two — maybe 20 or 30 people.

Where do you see proteomics headed?

I think how well the TAP technique can be used in higher eukaryotes will be a very interesting thing to observe. There was so much hype in proteomics, and now the big question is: Who is really delivering? For somebody working for a pharmaceutical company, the question is simply, ‘do we get good targets, or can we use these methods to actually isolate good candidates for drugs?’ My point of view as a scientist is, ‘who is really delivering to increase the understanding of biological systems?’ That is a very high hurdle for any technique, but this is the final measure for me. From my point of view, to generate understanding of biological systems simply by quantifying proteins is at least not an easy task, simply because proteins may be stored within cells in an inactive form. The majority of proteins may have double the activity if there is double the amount — that is absolutely possible — but then this must come out of the large-scale analysis. I haven’t seen too many convincing presentations going in this direction, I must say. Like many things in biology, reaching understanding [using proteomics] remains a difficult task. Large-scale techniques give you another point of view, but they don’t make the task necessarily easier.

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