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Alexander Kurosky on His NHLBI Center and Biodefense Proteomics

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At A Glance

Name: Alexander Kurosky

Position: Professor of human biological chemistry and genetics, University of Texas Medical Branch, Galveston, since 1975.

Director, NHLBI Proteomics Center, UTMB, since 2002.

Background: Instructor of human biological chemistry and genetics, UTMB, 1973-75.

Post-doc in molecular genetics, UTMB, 1972-73.

PhD in biochemistry, University of Toronto, 1972.

Research and development chemist, Canadian Breweries, Toronto, 1965-67.

BS in biochemistry, University of British Columbia, 1965.

 

How did you first get involved with proteomics?

I’m a protein guy — I got into proteins 30 years ago and have studied proteins in a variety of scenarios, focusing largely on blood proteins initially, and also focusing on proteolytic activity and protease structure and function. I did dabble for awhile with some of the bacterial toxins, in particular cholera toxin, and did some work with that. And then subsequently I got involved with looking at proteolytic processing phenomena, and what is still a fairly new area of protein processing.

Proteomics presented for the first time a new window to look at biological phenomena differently and globally. And that really struck me as being a very attractive, compelling area to pursue. The other issue is that I also was pursuing in a parallel fashion the development of mass spectrometry because I deal with a lot of analytical aspects of proteins and I could see that mass spectrometry was going to make a big impact on protein analysis. So I realized that — separately from proteomics, actually — and I was trying to put in place the concept of mass spectrometry [here at UTMB] because I felt for biomedical research, mass spectrometry had a lot to offer not only for proteomics, but for a lot of other purposes.

In what way were you pursuing mass spec before you related it to proteomics?

It offers an alternative method for obtaining sequence information, [and] it was offering an opportunity to validate peptide synthesis — so you synthesize a peptide and you can confirm that the peptides you make are in fact what they should be. Similarly, I also run a protein expression facility where we make proteins in yeast, and mass spectrometry offers us an opportunity to validate that those proteins are made correctly. So very accurate mass analysis clearly impacts a lot. And looking at post-translational modifications of proteins, mass spec is beginning to make some big inroads there.

So I pursued that, and I put in a couple grants here and there, and finally I did get an NCRR grant [about three years ago] to get a mass spectrometer and then I got the university to give some matching funding. And with that I put in place a mass spectrometry facility upon which I could build a proteomics effort. And I could add to it — some proteomics, 2D gel equipment and software and that sort of thing. And then very fortunately, we applied to the NHLBI and became one of the 10 proteomics centers.

What sort of mass specs did you start out with?

At the time, the area was, and still is, of course, developing at a rapid rate. When I first submitted the NCRR grant, the Finnigan ion traps were very capable-looking instruments, and I was focusing on [them] initially. But the NCRR review program is quite long, about a year. So in that year the Q-TOFs came out — the Micromass Q-TOF and the [ABI/MDS] Sciex Q-STAR instruments. And they clearly looked a little more capable than the ion traps, although they all have different pluses and minuses. But the TOFs of course were more money, so we did get the grant from the NCRR and then were able to get the university to add to it, so we purchased the Q-TOF too, and almost at the same time we also purchased an ABI Voyager-DE STR MALDI instrument. So they were pretty much state of the art at that time, and those two were able to launch us on the proteomics road. The Voyager is really the workhorse of proteomics — it allows us to identify proteins quite readily. It’s not as high-throughput as the new ABI TOF/TOFs, but it’s still a fairly high end instrument. [But] it suffers in that it has to be run manually, and I am now trying to find a way to get a TOF/TOF.

How has the research at your NHLBI proteomics center been going?

When you ask anybody how their research is going, the typical answer is, ‘slow.’ It never really goes as quickly as one wants. The first period really has been a major effort in getting the organization in place here at UTMB. Because it was fairly sudden — we didn’t have long-standing proteomics development here. But nobody does — the word proteomics doesn’t go back that far. So we did a lot of scrambling around to get things in place. There are lots of frustrations with that.

We’ve built our effort on seven teams. We have one team that I head — Allan Brasier is my associate director: He’s an MD and has longstanding interest and expertise in airway inflammation, and particularly in regards to respiratory syncytial virus, — [which is] kind of a cold virus prevalent with children, [with] implications that that could impact the later development of asthma. We have a focus here on airway inflammation, and when we constructed our proteomics initiative, we built it around that.

The team I head is a protein separations area — that involves 2D gels, tandem LC, mass spectrometry — all the protein separation aspects is sort of in my purview. Then we have a group here headed by David Gorenstein. David has been interested in the development of thioaptamers, and they are actually fairly well along on the development of thioaptamers for use in chip assays for specific proteins (see PM 8-1-03).

They have developed thioaptamers for NF-kappa B for example — it’s kind of a chemical recombinant library approach where they are able to select these aptamers that bind specifically to a given protein with a high degree of specificity, and then they can be used in a variety of ways, either clinically as inhibitors of specific proteins in some disease states assuming that they can get into cells — which I’m not sure is doable right now — [or] certainly for diagnostics they would be valuable. They have the advantage that they are much more resistant to degradation, and they’re easy to make, and they’re relatively cheap. So that group has already developed thioaptamers for AP-1 and NF-kappa B and they have a lot of strength in NMR so they’ve done the structures of these aptamers.

Collaborating with them is another team headed by Jim Leary, who has a lot of expertise in cell-sorting and cell separation methods and he is working in close collaboration with the Gorenstein group. The fourth group headed by Bruce Luxon is our informatics group. One of the regrets I have is that I wish we had put even more emphasis in bioinformatics initially than we did. Because the bioinformatics really sets the tone for your whole proteomics program because you have all that data coming in. We’re now in the process of hiring a PhD bioinformaticist to provide oversight for our proteomics bioinformatics. We hope to fill that position soon.

So the bioinformatics group is working on methods to analyze the data coming in from all the other groups?

Right. And it’s coordinating, putting everything in one place. When you do 2D gels, you have a variety of steps that happen and you analyze the gels by fairly sophisticated software, which in itself is not trivial, and then they have the mass spectrometry data, so you want to put everything in place where people can access it online readily. The other thing is that the software is not all in place yet. We are partnering with Nonlinear Dynamics, so we have put in several thousand of dollars in their software, and actually we did some of it prior to getting the NHLBI grant, because I thought at that time it’s important to have. So we are part of their development and we’re beta testing some of their software.

High-quality software can alleviate a lot of the issues and problems of 2D gels’ aberrations. [I think] 2D gels still offer the gold standard for comparing two scenarios and getting some information. The biggest criticism of 2D gels is that they tend to miss the low-abundance proteins, but we are developing strategies to enhance the performance of 2D gels so that we can in fact see these low-abundance proteins using what we call post-fractionation techniques — in other words, we develop a master gel, and then some of those very weak spots we can identify by doing fractionations on very high-speed methods using spin columns and then re-running gels and putting that data onto the master gel to identify those very weakly displayed spots, which initially when you’re dealing with the whole sample, you can’t identify by mass spectrometry. It also requires that you do have a very high quality software capability so that you can manipulate spots between plates and between different kinds of 2D gels.

So those four groups constitute the technology effort, and then the remaining three teams focus in on the biology, and these are divided up into the cellular model of airway inflammation and then the mouse model and human model. We’re focusing initially on our technology development and the cellular aspect with some mouse development and still very little human, and in time we would shift the emphasis to the more clinical areas, later in the [7-year] grant period. So we have moved along a little bit — we just submitted a manuscript [to the Journal of Virology], a large-group effort looking at respiratory syncytial virus effects on epithelial cells. It’s a 2D gel approach really to pick up some novel concepts of how respiratory syncytial virus affects A549 cells.

How are you applying proteomics to the biodefense efforts at UTMB?

With the interest in proteomics we were able to initiate some other proteomics developments on campus and there is an NIAID grant that I participated in that David Gorenstein is the PI on. That’s about $1 million per year for 5 years. That grant has major goals for developing proteomics capabilities to study potential bacterial and viral agents relating to bioterrorism. Getting the NHLBI grant was some leverage, I think, in getting that one.

Subsequently, we also participated in a major funding application for this RCE — the Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (see PM 8-1-03, 9-12-03). There were at least six centers funded. This is a $48 million effort headed by David Walker, who’s the PI. So one of the cores in there is a proteomics core, and we are putting that in place — it’s not fully in place yet. I’m just negotiating to hire a PhD bacteriologist, Laurie Sower, who is going to put her efforts onto developing and interfacing that proteomics initiative with other proteomics capabilities on campus, and also going out in the region and trying to integrate the regional needs for proteomics with our facility.

The RCE led to us obtaining the National Biocontainment Laboratory (see PM 10-3-03). So this whole concept of doing proteomics within a biocontainment area will be a new thing. We haven’t yet formulated how that’s going to happen. But we’re going to have to put equipment and proteomics technology within the biocontainment lab, so that’s going to be somewhat challenging. [I’m] looking forward to working with people and developing that capability.

 

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