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Deborah Ferrington on Aging, Oxidation, and Proteomics of the Eye

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At A Glance

Name: Deborah Ferrington

Position: Assistant professor of opthamology, University of Minnesota, since 1999.

Background: Post-doc in biochemistry (funded by American Heart Association), University of Kansas, 1997-99.

PhD in biochemistry, University of Kansas, 1997.

High school science teacher, 1980-83.

MEd in secondary science education, University of Pittsburgh, 1980.

BS in biological sciences, University of Pittsburgh, 1978.

 

How did you get involved in proteomics?

Back in my doctoral days, I was involved in a lab [where] we did a lot of tearing apart of the sarcoplasmic reticulum calcium ATPase, and trying to understand what amino acids were oxidized in aged muscle. We used digestion of the protein and then mass spectrometry to identify what the modified peptides were. It was what people call proteomics now, but it wasn’t called that then.

When I came here as an assistant professor, my research focus [was] trying to understand how proteins change with age, with a particular emphasis on oxidative damage to those proteins with aging. It goes along the lines of the free radical theory of aging. So with macular degeneration, the actual mechanism has still not been defined. The approach people have taken has been a more limited approach to looking at individual proteins or pathways or something — trying to understand what might be changing in the disease. Proteomics was the perfect application of a technology to trying to tease out what might be happening with macular degeneration. So that’s what [our recent NIH] grant was all about: using proteomics to look for specific patterns of either changes in protein expression, or changes in the oxidation state of proteins in donor eyes that we have evaluated. There’s a retina surgeon, Tim Olsen, who’s a collaborator on this project — he actually grades the level of degeneration in the donor eyes, and then we know exactly at what stage that donor was before they donated their eyes.

When did you come up with the idea for this project?

When I was first hired in the department, they wanted an expert on aging, but someone who could take a very fresh approach to understanding macular degeneration. I got very frustrated looking at the literature, because it was so limited in what we knew, and with my training in protein biochemistry, it was just the approach that I thought would work — this more global look at everything, rather than limiting myself to specific pathways that I would really have to guess about.

How far along in the project are you at this point?

Right now, I have a graduate student, Cheryl Ethan, [for whom] this is part of her dissertation topic, and she’s working on about three different manuscripts now. I also have one additional person hired to help with the hands-on in the laboratory. We’ve looked at the macula, which is the very center section that is most severely affected with macular degeneration, and we’re comparing it with the peripheral region of the retina. We’re looking at expression level changes, and we’re looking at also comparing 4-hydroxy-2-nonenyl-HNE, which is an oxidative modification that we can find with an antibody. So those are the things that [the student is] working on now, and she hopefully will submit a manuscript identifying the proteins that are modified by HNE in the next couple of months. So we’re kind of in the beginning, but not exactly in the beginning — we’ve got the details worked out but we’re pulling the data together right now.

What techniques are you using?

We’re doing this with 2D gels, normally trypsin digest, MALDI-TOF mass spectrometry, and then we do MS/MS sequencing — normally it’s with the MALDI — with the Q-TOF machine, but we’ve also done a little bit of electrospray. Also, we have a publication out about some work we’ve done in rat retina. That was just published in Biochemistry this past year. What we had done is looked in detail at alpha-a-crystalline in the retina. When we first found this, because it’s so highly modified, I thought, ‘Oh, we have contamination from the lens.’ But the more I looked into the literature, [I found] there is a fair amount of alpha-crystalline in the retina. So in this paper in Biochemistry, we actually have identified different sites of post-translational modifications using 2D gels and mass spectrometry — this combination of MALDI-TOF and electrospray.

[We use] a Q-STAR from Applied Biosystems. We have two different places where we go to use the mass spectrometers.

So you use a core lab at the University of Minnesota?

Yes. It’s really a fantastic core facility. We do all the sample preparation, 2D gels, trypsin digest, and preparing it for mass spectrometry, and I actually have three people in the lab now who run the machine. So we basically just use the machine and we pay by the hour.

What is the next step in the project?

The next question everyone brings up is — especially with the post-translational modifications, like oxidative modifications that we’re interested in — ‘Is it functionally significant?’ So now what we’ve really been working hard at is trying to identify the sites of modification. That has been much more challenging, especially with this HNE adduct, because [we’re] having a hard time seeing it without doing any kind of stabilization of the chemical moiety. It’s in low abundance in these 2D gels, it’s possibly not stable in the mass spectrometer, and maybe the peptide doesn’t fly well in the TOF with this lipid. So we’re kind of fighting that now. But really that’s the next question: ‘What’s the functional significance of these [modifications]?’ In order to answer that, we really have to know what the site is.

What techniques are you trying for this challenge?

We’ve tried a couple of different things with fairly limited success. One of our objectives is to take a whole homogenate from a retina, and then isolate the proteins that contain HNE, and we’ve tried immunoprecipitation using an HNE antibody. That’s been kind of marginal. And then doing a tryptic digest, and we’ve tried electrospray because we thought maybe it’s so different that we’d be able to see the HNE. We haven’t really gotten good results there. Now we’re working with another lab on campus that’s also interested in HNE-modified proteins. They’re doing chemical modification for the HNE and [we’re] seeing if we can immunoprecipitate using this chemical adduct to the HNE. So that’s one of the things — to try to increase the abundance of the HNE-modified peptides in our sample. I think we’ll have a better chance then of actually seeing it by mass spec.

What is your ultimate goal with this project?

If we could find common pathways that are affected during the disease process of macular degeneration, this would be a huge step forward in developing potential therapeutic interventions. Drug development is not my thing, but if somebody knows either the type of free radical that’s doing the damage, or a particular pathway that’s affected, it’s possible that drugs could then be developed that would provide some therapy that would slow down the progression of macular degeneration. To prevent it would be great too, but just understanding the mechanism is really important.

Is your funding entirely from the NIH?

No — I have a lot of other funding. The American Health Assistance Foundation gave me a grant this past year. There’s [also] been funding from the university graduate school, the American Federation for Aging Research, The Foundation Fighting Blindness, [and] the Minnesota Medical Foundation. In order to get an NIH grant, you have to basically have the experiments done. So I spent a lot of time when I first got here just getting these small awards, but they have really helped me build up my lab and get us going. The National Eye Institute is the NIH institute funding my current work with this.

Are you planning any other projects in proteomics?

Yes. We have a second area of work that ties in with the oxidized proteins and oxidative damage. It is some work we’re doing on the proteosome, which is an enzyme complex that’s responsible for degrading oxidized proteins. We’ve shown in a couple of different tissues, including the retina, that the proteosome activity decreases with age, and so it could be one of the reasons there are more oxidized proteins with aging. So we’re using proteomics to tear apart this protein complex to identify subunits of the complex that are modified by HNE and nitrotyrosine and so forth. We’re using proteomics technology in that respect where we have a really specific goal and a specific question and there’s one protein that we’re looking at.

For this [we’re also using] 2D gels, MALDI-TOF, and MS/MS.

What are some of the major obstacles or difficulties in proteomics that need to be overcome?

Probably the biggest one is trying to resolve membrane proteins. Especially in the retina there are a lot of membrane proteins I know we’re missing. We’ve done tricks like 1D gels, and we’ve gotten a little bit of information from that, but even with a gigantic 1D gel, it’s really difficult to get the kind of resolution you get with 2D gels. I think for anyone using a tissue — especially a tissue that’s highly membranous like the retina — that’s probably the biggest issue right now. There are a few things I’ve read about using LC to do separation instead of 2D gels, but we haven’t really looked at that much. Our hands are full with what we’re doing right now. But if somebody could come up with some magic solution where somebody could get membrane proteins to actually resolve on a 2D gel, that would be unbelievable.