AT A GLANCE
NAME: Alan Marshall
POSITION: Professor of Chemistry and Biochemistry, Florida State University;
Director, National High Magnetic Field Laboratory ICR program, since 1993
Professor of Chemistry and Biochemistry; director, Campus Chemical Instrument Center, Ohio State University, 1980-93
Faculty member, University of British Columbia, 1969-80
PhD in Physical Chemistry, Stanford University, 1970
BA in Chemistry, Northwestern University, Evanston, 1965
How did you get interested in FT-ICR mass spectrometry?
I started out in a six-year medical program at Northwestern University. I was supposed to go to undergraduate college for two years and to medical school for four years, but I hated medical school after the first year — too much memorization — so I switched to chemistry for the last year of my undergraduate program. Then I did a PhD at Stanford in physical chemistry, working with John Baldeschwieler. During that time, I was doing half NMR and half what I now do, ion cyclotron resonance.
After that, I went to British Columbia as a junior faculty member, and for the first five years or so I did NMR only. After I had been there for two years, I helped convince them to hire Mel Comisarow. He and I had overlapped for a year at Stanford; my last year as a grad student was his first year as a postdoc. He did ICR, and I did NMR, and we were separate for another three years or so. Then, in 1973, we got together to do Fourier transform ion cyclotron resonance, which was our innovation. The cyclotron goes back to [Ernest Orlando] Lawrence in 1929, but what we did was the Fourier transform aspect of it the way [Richard] Ernst had done it for NMR eight years earlier. You get the whole spectrum at once in the time it used to take to get one peak, so it made it several hundred times faster and also made the resolution up to 10,000 times better, which we realized as soon as it happened, but nobody had predicted that ahead of time.
In 1980, I went to Ohio State University to become a professor of chemistry and biochemistry, and director of the Campus Chemical Instrument Center. I stayed there for 13 years. In 1990 the National High Magnet Field Lab moved from MIT to Florida State University. I called up the director and said, ‘we also use magnets, is there a place for that at the magnet lab?’ So he invited about a dozen of us mass spectrometrists, and out of that came about three or four directions, one of whom was actually studying proteins. For the next two or three years, the director of the magnet lab and I went to DC and talked to NSF and NIH and DOE and asked for funding, and they all said no.
In 1993, NSF created a new grant program that was called Chemical Research Instrumentation Facilities, and that’s when I decided I should move. I came to Florida State in 1993 as professor of chemistry and biochemistry, and I am also director of the ion cyclotron resonance program at the National High Magnetic Field Lab. We applied for that NSF money, and we received about $1 million a year for the first five years, which then got renewed.
How are you equipped?
We started with a 9.4-Tesla magnet. Everything good in ICR scales with the magnetic field or the square of the magnetic field: The resolving power goes up, the highest mass you can go to does, the dynamic range does, the length of time you can keep an ion there to play with it does. That’s why I wanted to be here, because we had the biggest magnet. Since then, we have built up the facility with four permanent staff, a technician, a machinist, and about five postdocs and ten grad students at any given time. Last year, we had 117 visitors — we are a national user facility, and our charter is to develop the technique and make it available to people. Today, we have two 7-Tesla magnets, two 9.4-Tesla magnets, and we are building a spectrometer that will go with our latest magnet, which just got installed and appears to be working, a 14.5-Tesla magnet: that will be the highest field in the world. The highest magnet field of a complete commercial instrument is 12 Tesla, and those have only come out this year.
What can you do with these magnets, especially with regard to protein analysis?
If you think of the mass spectrometer as just a separation device, you can distinguish 100 times more components than with the best single-stage chromatograph — LC, GC, gels, any of those. But unlike the chromatograph, which only tells you that the peak is there, the mass spec tells you what it is if you can measure the mass accurately enough. What ICR does 100 times better than any other kind of mass analyzer is tell you what the mass is. Up to a mass of 1,000, it actually tells you all the atoms that are in the molecule. For bigger ones than that — for example peptides — you can tell which amino acids are there and whether they are modified or not. And if you do MS/MS then, you can tell where the sites of attachment of the posttranslational modifications are, as well as the sequence. The kind of problems for which we are the solution is when you have a complicated mixture. In fact, nowadays, we often don’t do separation beforehand; we just throw everything in the mass spec.
Can you give some examples?
One example is Alzheimer’s. We have collaborators in Sweden who do 2D gels of normal vs. Alzheimer’s patient samples. They stain those for sugars, so they know which ones are glycoproteins, and then they look for which ones are more abundant in the sick people than the normal. They send us those proteins, and we analyze them and tell which sugars are on which residue. This will be reported at the upcoming ASMS conference, and we have submitted it for publication.
Another example is determining the sites of protein-protein interactions. Suppose you have two proteins that stick together and you want to find out where their contact surface is. Together with collaborators at the University of Alabama at Birmingham, we studied a protein from the HIV virus which self-assembles to form hexamers that envelop the HIV virus. There is no NMR, even though the protein is 24 kilodaltons. The X-ray structures of the monomer and dimer are known, but electron micrographs show that they stick together six at a time. We compared the hexamer and the monomer, basically by spray-painting them. If you spray-paint an assembly and then pry it apart and look where the paint was, you know which parts were stuck. Our paint was D2O. We dropped the protein in D2O and it exchanged the amide hydrogens of the backbone. The faster they exchange, the more exposed they are to the solution. If we do monomer vs. hexamer and look for differences, we can tell which parts are stuck to which other parts. That allowed us to gain new knowledge of the way the protein capsid assembles. It was on the cover of the Journal of Molecular Biology this January.
What is the importance of protein analysis vs. other applications of FT-ICR in your research?
The other main thing we do is petroleum analysis. That’s the world’s other most complicated mixture. In a single crude oil sample we have found 30,000 compounds so far — we call it petroleomics.
What is the advantage of the hybrid FT-ICR mass spectrometers that have recently come out?
Electrospray is a continuous source; the ions come out all the time. FT-ICR is an inherently pulsed detector — you trap the ions, you play with them for a second, and then you are ready to start again. To make those two compatible, in 1998, we introduced an octopole — it could be a hexapole or a quadrupole — outside the ICR. We feed the ions in continuously and collect them, and then once a second we pulse them to the ICR. That allows you to get basically 100 percent duty cycle, so we don’t throw away the ions when we are not doing ICR. The vendors have pretty much all come around to doing that; IonSpec, Bruker, Finnigan now each sell versions of that. The commercial ones are either hexapoles or quadrupoles, but they are similar. The enhanced duty cycle is one reason to have an extra mass filter, or mass trap, in front. The second reason is, ICR has a limited dynamic range, and in biology, you often have a dynamic range up to 1010. The way out of that is, if you pass the ions on their way in through a quadrupole mass filter, then you can pick out just the ones you want.
Many proteomics researchers seem to be afraid of FTMS. Do you think this is going to change?
One of the reasons is, they are just afraid of the magnet, because they don’t do NMR, and if they didn’t do ICR already, they have to deal with a different kind of instrument. Then, the mythology has been that ICR is hard to do. I do it, and I have never thought that it was. But I think the performance difference is so high now, and the commercial machines have become a lot more user friendly, that people just cannot ignore it.
This year at Pittcon, [Thermo] Finnigan came out with its latest Q-ICR. It looks just like an LCQ, a quadrupole ion trap, as far as how you operate it, with just a couple of extra buttons that do the ICR. I think that will open the field to the people who used to be afraid, because now it just looks like their LCQ. [Thermo’s] pitch is that they want to compete with Q-TOFs, they hope to take a major share of that market.