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Researchers Look to Dendrimers as a Proteomics In Vivo Labeling Strategy


Andy Tao
Assistant professor
Purdue University
Who: Andy Tao
Position: Assistant professor, departments of chemistry, biochemistry, and medicinal chemistry and molecular pharmacology, Purdue University, 2005 to present; assistant professor, Burnham Institute for Medical Research
Background: PhD, Chemistry, Purdue University, 2001; postdoc Institute for Systems Biology, 2002-2004

Andy Tao and his colleagues at Purdue University are in the process of devising a method to label proteins in cells in vivo by bounding a dendrimer with a glowing identification tag. Dendrimers can pass through cells with little disturbance to the cells and then label specific proteins with isotopic tags while the cell is still alive.
If found to be effective, the technique would be useful in identifying and studying disease-causing proteins, Tao said.
Their research appears in Chemical Biology available here, and in Chemical Communications available here. 
Below is an edited version of a conversation ProteoMonitorhad with Tao this week.
Tell me what you did.
We certainly try to present a new strategy and a new quantitative reagent for quantitative proteomics analysis. There certainly are a number of quantitative reagents available today. We feel some of them, especially for small molecule-based reagents, after a level you have to remove them. Sometimes, you need an extra purification step to do that.
Some other groups developed these solid phase-based reagents. This type of strategy is better because it’s much easier to isolate and recover the peptide protein you want to label. The problem is that … solid phase extraction, I believe, is a heterogeneous reaction between your reagent and the targeted protein. So you have a lot of problems related to this heterogeneous reaction.
We addressed this problem by introducing a soluble polymer which means all the labeling, everything [is done] in the solution phase. And it’s completely homogeneous. Kinetically it’s completely homogeneous and linear.
The second question we addressed is potentially we will be able to do proteomics in vivo. I would say that 99 percent of proteomics, we do in vitro. And sometimes, you break the cell or destroy the tissue to look at a certain protein. Sometimes the natural condition is being broken.
Suppose, for example, you want to see the intact protein actually in the cytosolic version, but if you break the cell, maybe the nucleic portion comes out. And the physiological condition, you’ve destroyed.
We want to address this situation, basically, to do proteomics in living cells.
The reason we used this dendrimer-based polymer is it can penetrate the cell walls, and [then] we can label the protein or peptide in the cell and achieve quantitative proteomics.
What were the challenges in coming up with this technique?
The major challenge was that … in the beginning when we simplified these reagents, we wanted to characterize them, so we actually used a couple of techniques to do that, but we still feel that we cannot completely get good characterization of our reagents.
We developed several things to look at it, [including] a dye base to look at how many of the reactive groups we [could] put on the dendrimer and if the synthesis is successful. This is one challenge we saw in the beginning, but eventually we think all the syntheses worked out very well.
The second [challenge] is one we haven’t completely solved: How efficient, how good is it to do this in living cells? And we are able to efficiently deliver this reagent in the cells then take a look at it. We put a fluorescence tag on it so we could see how efficient it was.
We feel it was efficient, but the reaction and everything is still not characterized very well, and we see still … contaminated proteins coming out with our reactions. When you have a very high dynamic range, you always see some contaminated proteins.
What’s the significance of your research? What are the implications to proteomics research?
We provided a type of reagent and strategy for quantitative proteomics. And we feel this reagent can be better than existing reagents. [And second], the ultimate goal for us is to achieve proteomics in living cells. And on this, so far almost no one has demonstrated that.
Is that something that can be done now using your reagent and technique or is that goal for the future?
That’s a big objective that we are counting on doing. So far, we have demonstrated that it’s possible to deliver this reagent into the cell and then potentially intact into the protein. We then actually captured some proteins. But the targeting, everything, is much lower than we expected, so you would try to optimize the conditions, to increase the yield, to increase the efficiency.
What’s the major roadblock in trying to achieve that?
Dendrimer-based reagents for drug delivery have been worked on by multiple groups. But what is the effect of this? What’s the toxicity of dendrimers delivered into cells? It could disturb the whole cell system. Some groups working on this [have found that] sometimes the dendrimer is toxic to the cell, but sometimes it’s not, so there is no established report saying dendrimers have no problem or have problems.
Sometimes they have long-term effect and we don’t know. Certainly, there is disturbance to the living cell.
So this method, if we can achieve in vivo, in living cells, it would basically provide screening for drug targets.
Is that the major application for this method you’ve developed?
Yes, yes.
For personalized medicine?
Have you been able to observe anything yet with protein interactions that a proteomics researcher wouldn’t using other more standard methods?
In vivo is the ultimate goal, but I think we have probably a long way to go. We are pushing in that direction but so far, I still feel that quantitative proteomics, which means that we break the cell during quantitative analysis, I feel that with this type of reagent, it would work very well.
Why did you use snake venom in your research?
There are several reasons. First, snake venom has a high concentration of mixtures of proteins and other molecules. But the major reason is that snake venom has major pharmaceutical and medical implications. A lot of snake venom protein targets the neurological system and the cardiologic system.
Are there any plans to look at other types of fluids like blood or urine?
Oh yes, we consider this an improved version of ICAT. ICAT is popular for quantitative proteomics and for any biomarker discovery. This reagent can be used for all these quantitative purposes.

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