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NCI s Josip Blonder on Technologies and Challenges for Studying Membrane Proteins

Josip Blonder
Senior staff scientist
Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick

At A Glance

Name: Josip Blonder

Position: Senior staff scientist, Laboratory of Proteomics and Analytical Technologies, National Cancer Institute at Frederick, since 2002.

Background: Postdoc, Richard Smith's laboratory, Pacific Northwest National Laboratory, 2001-2002.

Physician specializing in cardiology in Libya, Bosnia and Herzegovina, and Germany, 1979-2000.

MD, University of Rijeka School of Medicine, 1978.

At this week's ASMS conference, Blonder gave a talk addressing some of the challenges of studying hydrophobic peptides and membrane proteins. ProteoMonitor caught up with Blonder to find out more about his background, and his technique for solubulizing membrane proteins.

What is your educational background and how did you get into proteomics?

I'm an MD and I got my MD degree in Croatia. I came to the USA five years ago, and I got a postdoctoral fellowship with Richard Smith. He's the head of PNNL's Laboratory of Micromolecular Biology and Mass Spectrometry. So there I finished a postdoc, and while I was working with Dick Smith, I was able to develop a method to solubulize membrane proteins using organic solvents, which is the opposite to what you typically use: detergents. The advantage of this approach is that I can solubulize protein in 60 percent methanol and digest them in the same solution. So this is actually single-step solubulization and digestion in buffered methanol. This platform had shown that it was pretty robust and very successful, so I have used this platform in conjunction with quantitative proteomics. I have selected O18 labeling, and it fits very well with the platform that I have developed.

This platform has been published in the Journal of Proteome Research, and I have used this platform to interrogate malignant melanoma plasma membrane. This is a collaboration. My collaborators have prepared the cells and the plasma membranes according to my instructions, and I have labeled them using O18, and we decided to profile hypoxic melanoma cells because it is well known that hypoxic stress really induces and selects for aggressive clones and more malignant melanoma.

What is the advantage of your method of membrane solubulization over other methods?

The majority of people use detergents to solubulize membrane proteins, and some people just shave the hydrophilic part of the membrane proteins and don't solubulize the protein. The main advantage of this method of using buffered methanol is that it is mass spectrometer friendly, because detergents really interfere with separation, which is upstream from the mass spectrometer, and they also interfere with ionization. This method is much more mass spectrometer friendly. And also, you don't need to use denaturants because organic solvents are good denaturants. So the advantage is that it makes sample preparation for mass spectrometry simpler and easier.

What got you interested in membrane proteins in particular?

When I came to Dick Smith's group as a postdoc, I was assigned to a project dealing with membrane proteomics, and since then this method has been successful, I was offered the job at NCI here where I still work on membrane proteomics. I like it because it's a tough, difficult, challenging part of proteomics to do because of the chemical heterogeneity of the membrane proteins. Also, 70 percent of the current pharmaceuticals really attack membrane proteins. So they are extremely important in cell biology, and in disease biology as well.

What particular projects were you working on in Dick Smith's lab, and at NCI when you first began here?

I was exploring the membrane proteome of Deinococcus radiodurans — it's a really tough organism that's resistant to many kinds of harsh environments.

After that I went to the National Cancer Institute's Laboratory of Proteomics and Analytical Technologies. Here I started working on Natural Killer cells. Basically I was trying to incorporate this method into multidimensional separations. In Dick Smith's group, I used just reverse phase LC to analyze it, and here I needed to apply this method to multidimensional proteomics and quantitative proteomics. So I first started with NK cells, then after that I was working on keratinocytes, exploring the plasma membrane of keratinocytes. Now we're working on analysis of HeLa cells.

You can say that one of the peak points in doing this is to look at the lipid rafts using this type of methodology in conjunction with multidimensional separations. The lipid rafts are actually the part of the plasma membrane — [they are] the tiny microstructures within the plasma membrane that are involved in signaling. So I was working to incorporate this method in multidimensional separation, and then to develop a method for mass spectrometry-based profiling of the lipid rafts. I was developing a quantitative approach to measure the lipid raft proteins.

The lipid raft is a scaffold, and the proteins are embedded there. So basically the thing is that you have to solubulize the lipid raft. Lipid rafts are detergent-insoluble, so I'm using my method to investigate the proteins which are co-purified within the lipid raft.

This is technology and methodology development, and later I applied this towards cancer research.

When did you start applying the technology toward cancer research?

So I came to this lab in 2002. The first year I spent optimizing all of this thing, and after 2003 and 2004, I was applying it to the cancer research.

What we've done here is, we have taken the same cell line of the malignant melanoma, and the control is just grown under normoxic conditions, while the one we would like to interrogate is grown under hypoxic conditions. Hypoxic conditions turn on different of genes and different types of signaling sequencing, and it is well known that hypoxia is really influencing the nature of malignant disease. So what I would like to do is do relative quantitation measurements between cells grown in normoxic and hypoxic conditions. We would like to see what gene products are up- or down-regulated when the cells are exposed to hypoxia, in order to elucidate putative drug targets which can be inhibited or down-regulated to stop malignant disease.

Are you studying the entire cell proteome, or only the plasma proteome?

Only the plasma proteome, because we believe that if we go to the more targeted proteome, we can more easily elucidate more-important and low-abundant proteins. This approach is called targeted proteomics, or subcellular fractionation.

Have you found significant plasma-membrane proteins related to hypoxia?

We have found the proteins which are upregulated and downregulated. Some of them are known suspects, and some of them should be further investigated.

Why are you looking at the plasma membrane in particular to investigate this type of melanoma?

It is well known that this type of cell line gives significant response to hypoxia. What we are doing now is global interrogation of the plasma membrane. So interrogating proteins one by one, we have been able to interrogate on a global scale. This is also known as shotgun proteomics, and we were able to identify 6,000 proteins using this technique. Validation will take definitely longer.

Hypoxia is related to all the cell metabolism, but the membrane is the first barrier which senses the hypoxic condition. Receptors in the plasma membrane will turn up or down different pathways, preparing cells to survive in hypoxic conditions. Only the cell clones within the tumor that are phenotypically able to survive hypoxia are multiplying, and they are more nasty and more aggressive. So the plasma membrane is the first interface in registering hypoxia and preparing the cells to survive hypoxic stress.

Do you think the proteins you have found will make for good therapeutic targets?

We are just in the beginning of validation. Of course, among them, some might be very good targets, but we still have not confirmed which would be good drug targets. Further validation is needed still on the biological side.

What projects are you planning for the future?

Well, we're going to validate this dataset and try to narrow down the important proteins involved in hypoxic response. Definitely, the ultimate goal would be … to interrogate not only animals, but also human specimens.

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