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UT Southwestern s Yingming Zhao on Studying Post-Translational Modifications

Yingming Zhao
Associate and assistant professor, department of biochemistry
University of Texas Southwestern Medical Center

At A Glance

Name: Yingming Zhao

Position: Associate and assistant professor, department of biochemistry, University of Texas Southwestern Medical Center, since 2000.

Background: Assistant professor, department of human genetics, Mount Sinai School of Medicine, 1998-2000.

Postdoc and graduate fellow, Brian Chait's laboratory, Rockefeller University, 1991-1998. PhD received 1997.

MS in bioorganic chemistry, New York University, 1991.

BS in physical chemistry, East China Institute of Chemical Technology, 1986.

Last week, Yingming Zhao gave a talk at the American Society for Mass Spectrometry conference about studying protein modification using a tagging-via-substrate approach. ProteoMonitor talked with Zhao to find out more about his approach, and about his research background.

How did you get into proteomics, and studying post-translational modifications in particular?

I got my PhD with Brian Chait at Rockefeller University. After that, I started my own research group. I was located originally at Mount Sinai Medical School in Manhattan. Then I relocated to the biochemistry department at UT Southwestern Medical Center in Dallas, Texas, about four and a half years ago. So, as a mass spectrometrist or protein biology researcher in the biochemistry department, you have a lot of colleagues whose main interest is in doing basic research. So identification of proteins and characterization of modifications for individual proteins is a basic part of the service or collaborations that we have occasionally.

We want to expand the mass spec tool for basic research. In biology research, most people are using a variety of screening tools to identify more protein candidates in response to disease, cellular environmental change, or whatever pathway. So in this kind of environment I just feel we need to develop more powerful proteomic screening tools so that we can quickly identify proteins involved in whatever pathway for cellular regulation, or characterize different biochemical processes.

What did you do while you were in Chait's lab?

In Brian Chait's lab, I did two things: One was to develop methodologies, another aspect was to apply those methodologies for protein identification. That was about seven or eight years ago, at the very infant age of proteomics. Now everything has changed.

A major bottleneck for efficient proteomic screening is high complexity and wide dynamic range of cellular proteins. We can not just identify all the proteins and leave [it] there. The key is to find proteins or biochemical processes that are relevant with cellular regulation and disease. So, although systems biology is kind of a holistic approach, in reality it's difficult to do 100 percent holistic. We still need to integrate reductionist approaches with holistic approaches to perform targeted proteomic analyses. Personally, I think that may be more efficient.

So, one way to do that is to target different modifications, to see what's significant, and what the dynamics of modifications are. Profiling of the modifications gives you some hint of the potential function of this modification and its regulation. Of course, protein-protein interaction is important. I got into protein modification research in Texas. We're also using protein purification in order to dissect pathways.

When did you start your work on the tagging-via-substrate approach?

About two and a half years ago. At that time, we thought [that] for some modifications, there's no good way to do it. So we thought about the Staudinger reaction. It's a reaction between an azide and phosphate. We thought that might be useful, so we tried that and it worked.

Basically, for any post-translational modification, one [reagent] is a protein substrate, another is a modification unit substrate. For phosphorylation, it's ATP; for protein farnesylation, it's farnesyl diphosphate. What we tried to do was put a handle on the substrate — it's an azide. It's small, uncharged, and it's pretty stable. Hopefully, the small azide in the substrate will not significantly change the structure of the substrate, so that the enzyme responsible for the post-translational modification reaction can still recognize the substrate. So the resulting post-translational modification will contain the azide. Then we can take advantage of the Staudinger reaction to put a probe to the azido-modified proteins. Then because we have a probe, we can detect these proteins. In such a way, we can detect and isolate the post-translationally modified proteins.

There are two requirements to make this happen: One, the substrate should be recognized by a natural enzyme. Two, the azido unit should be accessible to the phosphate.

Are there other technologies like this out there?

Well, this is a tag, and tagging is used all the time. Sumolation or ubiquitination is another way to do this. That's a kind of tag too.

We tested if it works for phosphorylation, farnesylation. I guess it works well for proteins that are reasonably hydrophilic.

Is the method patented?

We've filed a patent.

How does the method compare with some of the commercial options that are out there?

For farnesylation, there's really no good method out there. You cannot detect it unless you use radioisotopes. Farnesylation is required for activation and membrane association of Ras. The enzyme required for protein farnesylation is an anti-tumor drug target. For some of these clinical drugs, the true target is not Ras — it originally targeted Ras to block Ras' farnesylation, but in reality it's not Ras. So there must be something else that is responsible for that. If you have good proteomic technologies, you might be able to find the true target that is responsible for this clinical drug's effect, or anti-tumor activity.

What other projects are you working on?

We are very interested in IMAC (immobilized metal affinity chromatography) technology. We apply the technology to profile protein phosphorylation, and we are going to do quantification too. My personal bias is it looks like it works. At least in our hands, it works.

We are profiling adiposome proteins and mitochondrial phosphorous proteins. Adiposome's older name is lipid droplet. It's a subcellular compartment for storage of the lipid.

Did you find significant proteins using this method?

Yes, we found a bunch of key molecules involved in the regulation of adiposomes that are phosphorylated, but at this moment we still don't know its implication yet. That will require some biochemical work. But it gives some hint as to the key molecules involved.

Theoretically, if you can find some phosphorylation event that is responsible for mitochondrial function, it may give you some hint as to what kind of kinase is involved in that event.

What got you interested in adiposomes?

We really just provide proteomic support. It's really Dick Anderson's project. He's the chair of our cell biology department.

What are your plans for the future?

We have pretty good tools for phosphoproteomics. So I think we have pretty good screening tools. I think the next step is to apply the screening tools to interesting biological problems that are disease related, and then to characterize disease and understand disease. But we are in a very early stage [with] this.

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