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West Virginia University s Aaron Timperman on Seawater Proteomics

Aaron Timperman
Assistant professor
of chemistry
West Virginia University

At A Glance

Name: Aaron Timperman

Position: Assistant professor of chemistry, West Virginia University, since 1999.

Background: Postdoc, Ruedi Aebersold's laboratory of molecular biotechnology, University of Washington, 1997-1999.

Postdoc, Paula Coble's laboratory of marine chemistry, University of South Florida, 1995-1997.

PhD in analytical chemistry, University of Illinois, 1995.

Aaron Timperman was recently awarded a half-million dollar grant from the National Science Foundation for his project on "Seawater Proteomics" (see chart). ProteoMonitor caught up with Timperman to find out what seawater proteomics involves, and how he got into the uncommon field.

What is your research background? How did you end up combining proteomics with oceanography?

I went to grad school at the University of Illinois, and I did a lot of instrumental development there — capillary electrophoresis, and our group kind of focused on neuropeptides. So I was always interested in proteins, peptides. And then afterwards, I ended up going to the University of South Florida for two years. The first postdoc I did there I did in chemical oceanography. And I did that with Paula Coble. I'd done a lot of CE stuff before, so the whole thing that I was trying to do there was separate proteins from seawater by capillary electrophoresis.

The neuropeptide thing, I didn't really want to continue with that, and I was always just kind of fascinated by marine science too, so I decided to try that for a couple of years. So I did that, and I developed this capillary electrophoresis system. I left before I got a real good chance to put it to use. But at that point, I thought, well, if I can separate them with capillary electrophoresis and get a nice little signature with laser induced fluorescence detection, that only leaves me with a small part of the information. So at that point I really wanted to do the proteomics kind of thing. And then I had another postdoc with Ruedi Aebersold at the University of Washington.

What is the idea behind separating proteins from ocean water?

I have a big interest in molecular characterization of dissolved organic matter. The whole reason is we really don't know how dissolved organic matter cycles. So there's a lot of fundamental questions about dissolved organic matter cycling that really need to be answered. We know that we're putting a lot of carbon dioxide into the atmosphere, and other gases, and we really don't know how the earth as a climate system is going to change and to adjust to that. There's already ample evidence of global climate change, and dissolved organic matter is surely important in that cycle, because there's about as much carbon in dissolved organic matter in the ocean as there is total carbon in the atmosphere. The question then is how carbon from the atmosphere moves through the ocean — some of it is extremely stable and builds up to relatively high concentrations. What is it about those molecules, their structure, their environment that allows them to really accumulate? All those transformations of molecules as they go through dissolved organic matter are really unknown.

In the past, people have done a lot of 'class' sorts of analyses, looking at are there different reactive groups present, or if I treat it by such a method, what will happen to it? Our focus is much more at the molecular level. We know how to sequence proteins and peptides from gels and other things, so if we're going to look at dissolved organic matter at the molecular level, it makes the most sense to start with proteins, because they are so well characterized, and they're somewhat predictable as biopolymers.

If you really want to try to find out how dissolved proteins cycle, and you want to find out how dissolved organic matter in general cycles, you need to have some idea of what proteins are there, and how they change. It's hard to say how they change if you don't have any structural information at the molecular level.

When did you start working on characterizing proteins in ocean water?

A long time ago. I guess I started my postdoc in 1995, and it's been a slow and gradual progression since then. In fall of 1999 I started at West Virginia University, but it wasn't really until a year later that I was able to start that project here. Here I started the project in the fall of 2000.

How has the project progressed since 1995?

In 1995, at that point, we were just trying to separate things by capillary electrophoresis to get some sort of signature. So you would get some sort of electropherogram that you used laser induced fluorescence detection for. You would have these peaks, and you wouldn't know exactly what they were. But you could essentially compare them to each other and look for variability and whatnot, and look for signatures.

When I worked in Ruedi's group, I did some more instrumental development and learned how to do protein sequencing, but didn't do anything with seawater. So it wasn't until really some time in 2000 or 2001 that we were actually able to do some proteins in seawater. It was probably 2001 before we actually got some seawater collected for the proteomics project. Fortunately, my friends at the University of South Florida helped with that, because I'm not next to an ocean, right?

This is a long, drawn-out process, because first you've got to do all the gels and you've got to get all the sample preparation down and all that stuff. So it was probably around 2002 before we started doing the actual sequencing. We spent a lot of the initial time just doing methods, refining methods.

At this point, we have one paper that's just coming out in Marine Chemistry. It's on the web now. It's called "Marine Proteomics: Generation of Sequence Tags for Dissolved Proteins in Seawater Using Tandem Mass Spectrometry." That was published online last September. It's our first real report of doing this stuff. The interesting thing is that most of it is de novo sequencing, because there's very little that we've been able to match out of databases. It's kind of a unique problem that way because there are just so many organisms in seawater, and there hasn't been much genomic work that's been done on those. So most of the time we're doing de novo sequencing.

We've used some tools from, say, Finnegan to help speed things up. There's the DeNovoX program — we've used that quite a bit.

Once you've sequenced the proteins, how do you go about identifying their function and characterizing them in general?

That really opens up just a whole new field, because you can do a lot of homology searching, but of course, you're limited a little bit because we have relatively short sequence tags — they might be 9 to 12 amino acids. You're looking for homology across species. We've definitely gotten some hits and returns that make a lot of sense. One of the ones that makes the most sense is we found a sequence tag for rubisco, which is a photosynthetic protein — it's supposed to be the most abundant protein on earth. It makes sense that we've seen a peptide for that — I believe it's the first direct detection of it in seawater that's been reported. That's interesting, and it makes sense.

I think one thing that's a little more powerful, and a bit more reliable is that we see essentially three different classes of tags. The first class is non-specific tags. They're not unique sequences, and they don't really give us any sort of information. In another class, we see that the things that they match are mainly bacterial or envelope proteins. So they're these outer membrane proteins — there seems to be a preponderance of those. There also seems to be a preponderance of enzymes. The hypothesis at this point is maybe a lot of those enzymes are glycosylated and resistant to degradation. Membrane proteins are often rather stable.

In terms of seawater itself, the fact that [some dissolved proteins] are resistant to degredation tells us that enzymatic degredation is a really important process, and it's probably the controlling process that breaks down everything else really rapidly. And for some reason, there's a few of these things that just aren't easily digested by the enzymes in seawater.

The big thing overall is that there are people that try to model climate. The whole idea is to have good, experimentally validated information for them to plug into their climate models. If they know all the pathways into this dissolved organic matter pool, they know how it's controlled, and they know how things leave the dissolved organic matter pool, then they can actually incorporate that into their models. But we're not quite there yet. We've really just started.

Are there other people working on identifying proteins in seawater?

I haven't seen any other publications from any other groups at this point.

Where does the water that you sample come from?

Some of it comes from Hawaii, and some of it comes from Florida. In the future, most of it will come from Hawaii. The area in Hawaii has been sampled a lot, so there's a lot of ancillary oceanographic data that's been collected over the years — it's part of this HOTS program, which stands for Hawaiian Ocean Time Series. The University of Hawaii has a lot to do with it, because it's right off their site, and they do a lot of those studies. So there's a lot of information, and there's been some genomic work done there. That's really important, because hopefully we'll be able to do some database correlation.

There are three bodies of water there that are relatively easy to sample. You can get from the surface water to intermediate water, or what's actually called deep water, to bottom water. Those are distinct water masses, and they're relatively easy to sample without going that far off the coast.

We start out with 60 liters of seawater, and essentially we add a little detergent and do ultrafiltration. And of course, the whole thing that you're always concerned about is altering the sample. You want to get out all the particulate matter, the bacteria, the viruses and just look at the dissolved component. We end up precipitating it down at the end, resolubilizing, and running it out on a gel.

What do you hope to accomplish with the half-million-dollar NSF grant that was recently awarded to you?

The recent NSF grant is essentially a continuation of this work. We're going to keep on hammering on characterizing proteins in seawater for a while. We like to look at a lot of different types of water samples. We'd like to be able to extend our tag length. We'd like to be able to use different enzymes, and to search for modifications. There's lots of stuff to be done out there.

Do you think there are applications for those proteins that are resistant to degradation?

I don't know. I haven't really thought about that too much. But the thing to keep in mind is that "resistant to degradation" is environmentally specific. If you pull it out of that environment and put it in a different one, it doesn't necessarily mean it's going to be resistant to degradation.

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