Focus
Molecular Biology
Analytical chemistry thrives in a multinational
laboratorydevotedto molecular biology.
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molecular biology laboratory for generating cutting-edge science. might seem an odd place to find EMBL looks for the most promising analytical chemists, but there young scientists—group leaders should they can interact with the end users of their be European, but no such restrictions exinstrumentation. The European Molecular ist for postdoctoral appointments—and Biology Laboratory (EMBL—pronounced ensures a regular infusion of new talent by "Em-bull" by the locals) in Heidelberg, Ger- capping a normal stay at nine years. To many encourages cooperation between maintain some continuity, however, scientists working in molecular biology and EMBL reserves the option of a rolling teninstrumentation. ure system under which investigators sign three-year contracts after their original A brief history nine years are finished. Most scientists Conceived in 1962, EMBL was the brainadhere to the nine-year rule. child of Leo Szilard—the physicist-turnedKai Simons, program coordinator for biologist of atomic bomb fame. It was to cell biology and one of the most senior be a multinational molecular biology facili- staff members (he's been at EMBL since ty—which a single country could not eas1975), says that training independent reily afford—analogous to CERN. Molecular searchers has become the hallmark of biology, however, did not take the origiEMBL. The goal is to prepare scientists to nally anticipated path. Centralized reenter the difficult European university search facilities proved unnecessary for system, where scientists can be well into molecular biology, forcing EMBL to retheir 40s before they get the chance to think its mission. lead their own research group. Simons It found its calling as the training says that EMBL "profits from this 'defect' ground for young scientists and as a place in the European system." However,
Analytical Chemistry News & Features, November 1, 1997
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Meets Instrumentation
EMBL's unique structure makes it imperative that the laboratory's alumnifindjobs to show that the structure works. Everyone seems to agree that EMBL ii s "high-pressure environment", but the pressure doesn't resultfromthe need to please the administration. Unlike universities wherr "publish or perish" is something of a mantra, the pressure on EMBL researchers is selfimposed. Because they know from the start that they have to move on within a arescribed period of time, the young scientists feel compelled to make the most of their limited time. Matthias Mann, the mass spectrometrist who leads the peptide and protein group says "It makes everything fast moving You never start a project that will take e minimum of five years to complete" Despite the drive to succeed, competition between groups is rare. Mann says, "At other places, groups don't communicate. Here [at EMBL], people communicate and use each other's expertise. It makes things fast and efficient." Just what do chemists and physicists
do at a molecular biology lab? They creatt the tools that help answer biological ques tions. In doing so, they have to be firmly grounded in the biology; otherwise, why do it at EMBL? Instrument development is a vital aspect of EMBL's mission, and the biologists regularly collaborate with the chemists and physicists who compris< the biochemical instrumentation and cell biophysics (formerly known as the physical instrumentation) programs. Having instrument developers in close proximity to biologists helps the chemists and physicists see firsthand the real questions in biology. Simons says, "We had to learn how to live with the instrumentation scientists. We discovered that you couldn't just leave them alone and let them do their own thing. We had to embed them in the biology. We've taught them, and they've taught us." The MS gamble According to Wilhelm Ansorge, the program coordinator for the biochemical in-
strumentation program at EMBL, his suggestion to hire Mann was a gamble; it wasn't clear at that point how large a role MS was going to play in biological research. Clearly, the gamble has paid off as MS has begun to move into the mainstream of biological research. Although the protein and peptide group existed before Mann was selected, it was without the MS capabilities. Since then, much of the group's time has been spent developing MS methods for biological samples. For example, Mann and Matthias Wilm developed nanoelectrospray, a low-flow (—20 nL/min) electrospray technique that is particularly useful for peptide and protein analysis, where large quantities are rarely available, because a sample volume of 0.22 uL can last anywhere from 30 min to 2 h. (i). Even though small sample sizes can be analyzed regularly, it still can't be called an "easy" technique. A fine line
According to Simons, biologists and chemists are teaching each other.
Analytical Chemistry News & Features, November 1, 1997 6 7 3 A
Focus
Ansorge develops large-scale DNA sequencing techniques.
often lies between having barely enough and having an excess. JVlann agrees with Simon s statement about chemists needing to know the biology. "In a chemistry or physics department, you [could be] trapped into working on a single technique that may or may not be relevant. You can only develop methods relevant to modern biology if you're in contact with the real biology and have the resources for instrument development." The challenge for Mann's group has been in convincing biologists that MS can answer their questions. Mann has collaborated with biologists to help sequence some of biology's most elusive proteins. "Our tactic has been to work on high-profile cases, such as apoptosis (or cell death)," he says. "Thaf s the only way to convince biologists." Collaborating with scientists at the Germany Cancer Center in Heidelberg and the University of Michigan, they used tandem MS to sequence FLICE, an elusive 55-kDa protein involved in receptormediated apoptosis (2). Mann's group also participated in the partial sequencing of one of the proteins in the telomerase of Euplotes aediculatus, a ciliated protozoan. Telomerase is a ribonucleoprotein that is responsible for the replication of the telomeres at the ends of chromosomes, which could be important in cancer and aging studies (3). Cloning of this telomerase led directly to the identification oo fuman telomerase (and MS thus made front-page news in the Herald Tribune, ,ays Mann). 674 A
MS technology may be answering important biological questions, but room for refinements exists. Mann says that although protein identity verification is now routine, a 10-fold improvement in sensitivity is necessary before routine sequencing of unknown proteins (so-called "de novo" sequencing) will be possible. Mann's group, in collaboration with Kenneth Standing's group at the University of Manitoba (Canada) and PE Sciex, recently demonstrated that the combination of nanoelectrospray, isotopic labeling, and quadrupole/iime-of-flight MS makes such de novo sequencing possible (4). Further refinements in the software for protein database searches are still necessary. As the MS technology matures, the peptide and protein group is starting its own biological projects so that it can do a
EMBL embeds chemists and physicists in biology and lets them develop relevant instrumentation. little more than strict instrumental development. However, Mann realizes that many areas of biology are so fast moving that it's difficult to compete, particularly for newcomers trained in other fields. Although the group has until now participated in extremely high-profile cases to help "sell the [MS] story", they are trying tofindan "in-between" area for their own biological research. To that end, three "real" molecular biologists have joined the group.
large-scale genomic sequencing. In fact, they did much of the design work for Pharmacia Biotech's DNA sequencing system. The group is currently working on the next generation of DNA gel-based sequencers. This system uses four lasers, four orfivedyes, and an array detector. Each reaction allows the sequencing of 3000-5000 bases (-1000 bases per lane). "We test the technology," says Ansorge, "but industry can do large-scale sequencing better than government centers." The two microscopy groups, which concentrate on scanning-probe methods and light microscopy, are part of the cell biophysics program. J.K.H. Hbrber's group applies various scanning-probe microscopy techniques to biological problems to understand physical properties. For example, the group used images of bacteriophage T5 tails to study the electron tunneling process through protein structures (5). Another research problem involves mechanical (or stretch-activated) ion channels in membranes that are not well understood. By combining scanning-force microscopy (SFM) and the patch-clamp technique, Hbrber's group can simultaneously control the electrical and mechanical properties of membranes. This ability allows the study of these channels. They have investigated membrane vesicles from Xenopus socytes as well as the piezo properties of hair cells in the inner ear (6) The light microscopy group led by Ernst Stelzer concentrates on "designing light microscopes especially for biological
Microscopes, t w e e z e r s , and other tools
Ansorge, in addition to his role as coordinator of the biochemical instrumentation program, serves as group leader for the microanalytical techniques group, which focuses almost exclusively on DNA methods. The group develops methods for
Analytical Chemistry News & Features, November 1, 1997
Mann is "selling the MS story".
research," according to Steffen Iindek, a postdoctoral associate with the group. The group designed a confocal theta fluorescence microscope in which the illumination and collection objectives are orthogonal to one another, minimizing the observation volume and improving both the axial and spatial resolutions. By incorporating mirrors in the optical system, theta microscopy can actually be achieved with a single objective. The newest member of the family of microscopic techniques at EMBL—jointly developed by the scanning-probe and advanced light microscopy groups—is photonic-force microscopy, which combines laser tweezers and two-photon absorption. The laser tweezers capture a 200-nm fluorescent latex bead, which is moved across biological samples. The microscope is limited to forces below 10 pN otherwise everything will be "cooked". The method has been used to image specimens such as rat hippocampal tissue; it was designed for nanometer-scale investigations of molecular interactions determinations of membrane structures and regulation of active intracellular transport The instrumental development groups are only a small portion of EMBL. However, these groups serve the important function of ensuring that the molecular biologists have the tools they need to answer their questions. Simons observes that the instrumental projects are driven by biology and not simply the desire to build a newer instrument. **Why compete with industry when they can do it better? Only by working on the interface do we have a chance to compete." Celia Henry
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References (1) Wilm, M.; Mann, U.Anal. Chem. .996, 68,1-8. (2) Muzio, M.; Chinnaiyan, A M.; Kischkel, F. C; O'Rourke, K;; Shevchenko, A;; Ni, J.; Scaffidi, C; Bretz, J. D;; Zhang, M;; Gentz, R; Mann, M.; Krammer, P. H;; Peter, M. E.; Dixit, V. M. Celll996,85,817-27. (3) Lingner, J;; Hughes, T. R; Shevchenko, A.; Mann, M.; Lundblad, V.; Cech, T. R Science 1997,276,561-67. (4) Shevchenko, A; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997,11,1015-24. (5) Gu6nebaut, V; Maaloum, M.; Bonhivers, M.; Wepf, R; Leonard, K.; Horber, J.K.H. Ultramicroscopy, in press. (6) Mosbacher, J;; Haberle, W.; Horber, J.KH. /. Vac. Sci. Technol. B1996,14,1449-522
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