At A Glance
Name: R. Graham Cooks
Position: Professor of chemistry, Purdue University, since 1971.
Senior Investigator, Inproteo
Background: Published recent paper in Science describing a method of making protein chips entitled, “Preparing Protein Microarrays by Soft-Landing of Mass-Selected Ions,” Science. 2003 Aug. 14.
Fulbright Fellow, University of Warwick, 1981.
Assistant professor, Kansas State University, 1968-71.
PhD in organic chemistry, Cambridge University, 1967.
PhD in organic chemistry, University of Natal, South Africa, 1965.
BS in chemistry, University of Natal, South Africa, 1961.
How did you get involved with proteomics?
I’ve been in mass spectrometry my whole career. I was involved early on in the development of tandem mass spectrometry for mixture analysis. We built, arguably, the first tandem MS/MS instrument in the early 1970s.
Tell me more about building the first tandem MS/MS …
It was built here starting in ‘71 in order to be able to look at individual ions in a mixture of ions. Chemical ionization was just being developed at that time, so we put a chemical ionization source on it and then did CI so as to convert each molecule in a mixture into the protonated form of that molecule, and then separated out those ions — so we had done the first stage of the two-stage MS/MS experiment. Then the separated ions were dissociated by collision, which is still the standard method. My background was in natural products organic chemistry, so a lot of the work we had done in those early years was complex natural products mixtures. Our modus operandi in that time was to do no chromatography and that remains true. So we did complex biological mixtures with low molecular-weight compounds and we were interested in identification and quantification of those compounds.
How did you get into proteins from this background?
For a long time, I’ve been interested in ionization methods — going back to the mid-’70s I was interested in SIMS — secondary ion mass spectrometry — for analysis of biological compounds out of complex matrices to couple with these tandem mass spec methods. I did some peptide mass spec in the late ‘80s and early ‘90s, so to some extent [the protein interest] came from there, but it really came from the establishment of the Indiana Proteomics Consortium. I was one of the people who was pushing for a strong instrumentation-based interaction involving both Purdue and Indiana University because of the strength of both places in analytical chemistry, which is my discipline. The people who were involved at that time were Richard DiMarchi — a former executive at Lilly and now at Indiana [University] as of just a couple months ago —, and our president Martin Jischke, and Myles Brand, the [former] Indiana president.
My involvement came about as a result of a slightly earlier institution called the Indiana Instrumentation Institute, on which I’m a PI and which was funded by the State of Indiana 21st Century Fund. The concept there was to increase our abilities and to have some effect on employment by developing an instrumentation-based research interaction involving the two universities and various state industries, and Lilly was involved with that. The two universities are generally ranked in the top three in analytical chemistry in the country. The idea of using and extending that expertise was the basis for the institute, III.
I think Jischke, as much as anybody, was responsible for directing it towards proteomics. We decided to build instrumentation in order to do some protein mass spec, but of course in this late stage in the game, you don’t just build a mass spec and take mass spectra of proteins. And so I went back to something that I first published in the ‘70s, which is a soft-landing experiment. What we set out to do and published in ‘77 is that you can separate ions with a mass spec and you could perhaps put those ions down on a surface. We published in Science in ‘97 showing the landing of organic ions into a fluorinated self-assembled monolayer — so an inert surface. The importance of that experiment was two-fold: We were able to demonstrate that it was possible to land the ion intact without breaking it, but more importantly perhaps, we were able to demonstrate that some ions could be landed and retain their charged character. So those ions could then be taken out of a vacuum system and even walked around in air without losing their charge or at least without completely losing their charges.
So how do you do the soft-landing?
It’s not a hard experiment. Everyone knows how to ionize anything basically, and so having ionized, a mass spec is nothing but a separator. And having ionized and separated, you can take that information and run, which of course is what is usually done. Or you can use the physical material that you have, which is a separated biological molecule that just happens to be in the charged state. That’s what we decided to do — just collect this material. Of course proteins are denatured on certain surfaces so we don’t want to use those surfaces. It turns out what we think are the best surfaces are liquid. So the protein is being put back into the kind of material that it first came from. We use hydrophilic but obviously relatively viscous and nonvolatile liquids, otherwise we couldn’t put them in the mass spectrometer. Then it’s just a matter of adjusting the energy.
What is the advantage of creating a protein chip this way?
The advantage is relatively quick access to pure compounds out of a complex mixture. So if you have a limited amount of material — serum or whatever — and you run a mass spectrum and you become interested in a certain constituent, you can go gather more material and do the standard workup followed by chromatography and whatever. Or else you can do this experiment where you’re in the mass spec already, so you take a look at this material by some standard biological assay — put it down, pull the chip out of the mass spec and test it — and those tests are then standard tests.
Can you give me an example of how these chips can be applied?
They could be applied, for example, to testing for proteins in serum that are going to interact with a particular drug candidate. You would treat each well in the plate with a drug candidate and look for binding. You could look in the standard ways, or you could do that experiment in situ and use mass spec to see whether you’d done the binding. The other thing that it could be used for is for purification — so if you would like to know if it’s worth proceeding with a particular protein, this is a purification procedure which will give you a small amount of material, and then you can decide on the basis of some initial assays whether to go forward.
Tell me about how Thermo Finnigan and Inproteo are involved with all of this.
This is a device that would be an attachment to a prototype mass spec from Thermo Finnigan. It’s easy to see that this could be attached to all sorts of Thermo Finnigan mass spectrometers. Thermo Finnigan supports the work in my lab. I’ve had a relationship with them — a research support sort of relationship — for many years.
People might want to explore [the chip-mass spec interface] as additional hardware on a mass spec — just like different types of ion sources have been used to allow new experiments. This is a back-end rather than a front-end addition. That’s the commercial value.
Inproteo funded this entire project, and they can commercialize the results. This experiment was part of the original proposal when we were setting up Inproteo — there were a number of investigators and each person had a particular project. This was my project.
We’ve done this experiment on two kinds of mass specs: One was a commercial Thermo Finnigan quadrupole instrument that we modified. It took us six months or so before we had that done and some data coming out of that. And then we, in collaboration with Thermo Finnigan, were able to build their linear ion trap into a self-standing instrument. We needed a surface and the ability to move that surface relative to the ion beam in order to be able to put material down at different positions. We needed to be able to introduce that surface into the vacuum system and take it out of the vacuum system — so what I’m calling the back end is part of a vacuum system with a gate valve and the ability to transfer this plate — where you can put material down at different positions and, under vacuum, move that plate around.
Are the mass spec modifications going to be commercialized as well?
That would make sense.
How do you all work together in the Inproteo group?
My weak point is [that] I’m not a protein biochemist. The people who are strong on the applications are our partners at Eli Lilly — John Hale, Mike Kneuman, and Rick Ludwig — so they’ve really been extraordinarily helpful in directing us toward interesting problems.
What else are you working on?
There are lot of other things we do. We’re building miniature mass specs for environmental and chemical agent monitoring — tiny MS/MS mass specs. Currently, you can put it on your shoulder and carry it around, including all the power supplies and electronics. We hope in the next year to get it down to 1 kg. Then you can do fieldable analysis. You don’t bring the sample back to the lab, you take the mass spec to wherever you’re going. Homeland security is a big application.