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Robert Cotter on the Development of Mass Specs, Including a Mini-MS


At A Glance

Name: Robert Cotter

Position: Professor of biophysics and biophysical chemistry, Johns Hopkins University

Background: Professor and research associate in pharmacology and biophysics departments at Johns Hopkins University, since 1976.

Assistant professor, Gettysburg College, 1973 - 1976.

PhD in physical chemistry at Johns Hopkins completed in 1972.


What was some of your early work in mass spectrometry?

I was in graduate school in the 1970’s, involved in the physics side of mass spectrometry. I originally had a background in physical chemistry. In grad school, I was working on reactions that take place in mass spectrometers in the gas phase. The first key to it is that the inside of a mass spectrometer is a vacuum. So there’s no air for things to collide into. That’s important in order get a mass spectrometer to work because in general one produces an ion and then accelerates it somehow and then uses either the velocity or the momentum to distinguish the mass.

So most of the instruments these days are time of flight mass spectrometers, and the idea is that you accelerate these ions to the same energy, but they all have different velocities and so the time of flight of every mass will be different. If it were to collide with anything, then all bets would be off because that would slow things down, so it’s important that it’s a vacuum. But you can purposely cause a collision between an ion and a molecule by inserting the molecules at the right point. Then you have what’s called a tandem. So the ions go through the first mass spectrometer completely in a vacuum. They then collide with a gas, and the result of them are recorded. That was something that was much newer in the 1970’s. It was used mostly for small molecules. I looked at reactions of water ions actually, at that time. But [tandem mass spectrometers] have gotten resurrected in more recent years because of the interest in biological structure.

When did you get involved in developing mass spectrometers?

After I came back from teaching in Gettysburg [Pa.], I got involved in developing new mass spectrometers. What I started off with was a laser desorption mass spectrometer. My expertise has always been on time of flights. Every mass spectrometer has an ionization technique — something that makes the ion charged, and then a way of then measuring the mass. So for example one of the ways of making the ions is called laser desporption, and then time of flight is the method of measuring mass. So I put together a laser desporption/time of flight mass spectrometer. I was one of the earliest people to do laser desorption before the more famous MALDI came about. So we were using infrared laser desorption, and we were doing structural problems. We looked at some small peptides — not very large and we looked at a lot of bacterial glycolipids using that technique.

How did you get the idea to use infrared laser in order to create ions?

That was kind of an interesting thing because when I first came here we got started on the idea of using rapid heating to make ions, and in fact we were able to get things that normally one would expect to burn up in the mass spectrometer to decompose by doing very rapid heating actually make ions. So the next logical step was to use infrared laser which has very rapid heating. That was the focus of the time.

What were some of the technical difficulties of developing that machine?

It’s always a little bit strange to do instrument development in medical school because they don’t have the same complement of machine shops, but we were lucky and did find machine shop to do this work. We as much as possible tried to use commercial instruments instead of building everything on our own — not reinvent the wheel — so I did in fact buy a time of flight mass spectrometer, and I modified it.

What other types of mass spectrometers have you built?

One of our big areas right now is building miniaturized mass spectrometers. And the motivation for that, or at least the funding from it, is coming in part from people who are interested in bioagent detection, or homeland security. We’ve developed a miniature mass spec with a three-inch mass analyzer and we’re able to have a mass range of up to 130 kilodaltons. The instrument as a whole is smaller than the size of a desktop printer. On the medical side, the concept is the following: Sometimes people talk about three stages in proteomics. One is the discovery phase, when one wants to find out the difference in a patient that has cancer and one that doesn’t have cancer. Then what we call the validation stage is the one that requires the most expensive mass spectrometers in trying to figure out when the peak is different, what it is — if it has any meaning to that disease. Not everybody does that, but I believe it’s a necessary step, because you could be defining a biomarker that’s really not specific to that disease. And then the third step, once you know the biomarkers is that you could use a miniaturized instrument that will eventually be a point of care mass spectrometer.

So that’s something that could eventually be used in a clinic?

Yes, that’s right.

What made you interested in getting involved in that last phase of proteomics?

Well we’re probably in two phases, but our interest over the years in designing mass spectrometers has been to determine structures. Not just to do diagnoses but to determine the structures of things. Most of the work we do is structural biology. We’re working with glycolipids, various proteins or enzymes that we try to identify. So the interest has always been in identification. We had a fairly big program here of looking at MHC antigens to try to identify what they are, and that uses a mass spectrometer. And in the course of doing that, we’ve become interested again in tandem mass spectrometry. There are two commercial instruments out there now, and we’ve designed another one.

ABI and Bruker both market tandem/TOF mass spectrometers. We’ve had a link for a number of years with Kratos Analyticals from England. They make an instrument that uses technology developed in my lab, and we’ve now bumped that up to a tandem, which we think is better.

What is the relationship between looking at MHC antigens and using a tandem MS?

The MHC class I peptides are all nine amino acids, so they fall in the mass range of 900 to 1300 — that’s only 400 mass units, but yet there are thousands of [MHC peptides], so a lot of them have the same mass, so the only way to really sort that out is to then break them up and find out what the structure is. So tandem is essential in that case.

Are you currently working on developing a tandem mass spec?

About 12 years ago we built a tandem mass spectrometer and we published it and we eventually abandoned it partly because when we tried to renew the NIH grant for it, they didn’t think it was worthwhile doing — I always have a saying “No idea before its time”. But because people have gotten interested in that again, we have worked with Kratos to convert their instrument into a tandem. Their instrument has all the necessary parts to operate as a tandem and we really only needed to add a collision cell. What we developed here was a thing known as a curved field reflectron. The time of flight mass spectrometer is basically a long tube. You give all the ions of all masses the same energy, and you do it within a few nanoseconds, so you start them all down this racetrack basically at the same time. The lighter ones move faster than the heavier ones, and you measure their time of flight. But it turns out the energies that these ions have is not just the energy you give them, but there’s some thermal energy because they’re an ensemble of molecules that have different energies. And that sort-of destroys the resolution. What you can do is as the ions get toward the end of the tube, put a reflecting field and send them back again.

So the ones that are moving a little bit too fast go a little further into that field, then go back and catch up with the others. So I have analogy for a reflector — I remember years ago when I was in summer camp we had a counselor that used to have us run races. He would tell all the kids to run out into the field, and then at some point he’d say “turn around,” and even the little kids got back at the same time, because the bigger kids had run too far. That’s what happens here. Everything catches up and the resolution sharpens right up. So what we did is we built a reflectron with a non-linear field which we call the curved field. And what it does is it takes care of things that come apart on the way down the mass spectrometer and refocuses those so you can actually get the amino acid sequence. If what we send down is a peptide, and it came apart on the way down, then when they return, they’ll return at slightly different times and you have a set of ions that represent the amino acid sequence. The curved field reflectron enables you to get sequencing. This reflectron has been in the Kratos instruments for about eight years and its doing very well.

What are you working on developing for the future?

I think what I’m really interested in is this miniaturization. My ultimate goal is that it would be a point-of-care instrument.

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