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Cheng S. Lee On Proteomics Technologies Being Developed at Calibrant Biosystems

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Cheng S. Lee
Associate professor, department of chemistry and biochemistry
University of Maryland

At A Glance

Name: Cheng S. Lee

Position: Associate professor, department of chemistry and biochemistry, University of Maryland, since 1998.

Background: Co-founder of Calibrant Biosystems in 2001. Member of company's scientific advisory board.

Assistant professor, Iowa State University, 1993-1997.

Assistant professor, University of Maryland Baltimore County, 1989-1993.

PhD in chemical engineering, Rensselaer Polytechnic Institute, 1988.


Cheng Lee, a co-founder of Calibrant Biosystems, was recently awarded a $273,000 grant from the NIH to study cell death using top-down and bottom-up proteomics. ProteoMonitor talked to Lee to find out more about his background, the apoptosis project, and about the work going on at Calibrant.

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

I actually came from a PhD in chemical engineering. When I started my first academic appointment at University of Maryland Baltimore County, that's when I started to get involved in the instrumentation development of capillary electrophoresis. Interestingly, that project originated from a research collaboration with a retired electrical engineer. His name is Bill Blanchard. He was in the area looking for the academic researchers who were interested in capillary electrophoresis, so we started working together. The project involved the development of instrumentation methods, instead of chemical methods, to control the electro-osmosis flow in electrophoresis.

During that period of time, we not only got a patent together, but we also attracted the attention of Hewlett Packard and later Agilent. So we licensed the first patent to Hewlett Packard and worked with them during the early stage in the development of this instrumentation feature — the control of electro-osmosis flow in electrophoresis.

What is the application for this?

If you can imagine changing the flow during separation, that would allow the proteins and DNA to resolve better inside the capillaries. So you can pretty much optimize the separation resolution together with the analysis time.

What did you do after you developed that technology?

Around 1992, while I was still at UMBC, the professor Daniel Wong — he was the center director for the biotechnology process engineering center at MIT — he came to UMBC for a sabbatical leave. At that time, the center was heavily involved with the characterization of recombinant proteins, particularly in the characterization of glycosylation patterns in glycoproteins. So I started working with the center from 1994 to 1997. That's how I got into the area of bioanalytical chemistry.

In the mean time, I moved from UMBC to Iowa State University, the department of chemistry. Iowa State University has one of the best analytical chemistry programs in the country. So from there, I developed the interest and the technology to couple capillary electrophoresis with mass spectrometry. Maybe that gets into why I finally got into the proteome area.

Around 1997, my student and I developed the combination of capillary isoelectric focusing with mass spectrometry. Capillary isoelectric focusing is one of the separation modes in capillary electrophoresis. It has the best resolving power particularly for proteins and peptides. Back then, most people thought it was close to impossible to combine CIEF with mass spectrometry through the electrospray ionization interface, because the CIEF used carrier ampholytes to create a pH gradient inside the capillary. So in order to separate proteins based on their differences in isoelectric point, you have to have carrier ampholytes in the capillary together with the sample to create a pH gradient. And the carrier ampholytes are small polyelectrolytes — highly charged small molecules — so they compete with proteins for electrospray ionization.

But then we were able to optimize both the ESI condition and the CIEF separation technique, and we were able to couple these two very powerful techniques together. Because of that, we started a collaboration with Richard Smith at Pacific Northwest National Laboratories in 1997. As you know, Dick is very interested in both LC and also capillary electrophoresis coupled with mass spec. We collaborated for a period of two to three years, and because of this collaboration, I became one of the co-PIs in one of Dick's early proteome projects funded by the National Cancer Institute.

That happened around the same time I moved back to the University of Maryland, this time at the main campus in College Park.

What are you currently working on? Can you describe your recently funded project entitled 'Unraveling Cell Death via Top-Down/Bottom-Up Proteomics'?

In our recent work, we've been working with collaborators at Yale and NIH, studying the proteome of tissue samples. The amount of protein from these microdissected tissue samples is very small — typically one microgram to 10 or 20 micrograms. For tissue-based clinical proteome studies, our technique becomes quite unique in providing not only the sensitivity needed but also the ability to handle the small amount of samples.

The NIH-funded project on cell death is actually one of many projects. NIGMS funds the fundamental biomedical sciences, and therefore in this current project, we are not only using the CIEF, but also we are developing a capillary gel electrophoresis, which will give us the ability to resolve proteins and peptides based on their differences in molecular weight. Both PI and molecular weight — these are very important for the confirmation of protein identity, and it will give us additional information towards the post-translational modification analysis.

In this particular project, we are going to use capillary gel electrophoresis as the first separation dimension, followed by the second separation dimension, which is reverse phase.

How does that relate to cell death?

There are two objectives in the project — one is technology development. The other is using this technology to study apoptosis and autophage cell death, using Drosophila as the model system. This is another collaboration with Eric Baehrecke at the University of Maryland Biotechnology Institute.

We are using two different model systems from Drosophila — one is embryos, and the other is from salivary glands. With the variety of mutants available in Dr. Baehrecke's laboratory, we can study the protein profiles of the embryos during development, and also salivary glands during the induction of the programmed cell death. We can introduce a steroid at different time points to induce cell death.

This project was started April 1 of this year. Understanding programmed cell death can certainly lead to the possibility of drug discovery and also cancer therapy, etc. It's also a more fundamental project that fits very well to serve the interest of our research.

When did you co-found Calibrant?

Calibrant was officially funded by late 2001. In addition to myself, another founder is Professor Don DeVoe from the University of Maryland College Park. Don and I had collaborated in a variety of microfluidic projects. We were interested in developing high-throughput protein separation techniques using disposable plastic microfluidic chips.

An analogy is to transform the current 2D PAGE into a chip-based technology. The advantages is you can separate much faster, and you can have automation, and consume much less sample. If you look at the current proteomic front-end technologies, including 2D PAGE, and multi-dimensional capillary techniques or LC techniques, it takes a day or two days, at least, for the analysis of a single protein sample, plus a lot of time for data analysis. Our thinking is that that's just not going to be high-throughput enough for biomarker discovery. If we can reproducibly and sensitively generate the protein profile — the protein patterns — just like you're getting from the 2D PAGE — if we can get that done in 5 to 10 minutes, then you can screen a large number of normal versus diseased samples. That will allow you to — down the road — validate what those proteins are.

There are just not many technologies today that will not only give you high-throughput protein analysis, but also display thousands of protein spots that are real, that carry physical characteristics such as PI and molecular weight.

So that's the platform of Calibrant — we call it Orion.

What other projects are you working on?

Well we began with the discussion of the electrokinetic-based multidimensional protein/peptide separation technology. That's the platform also under development by Calibrant, called Gemini. And right now we are also developing a technology called Cepheus. The current technology with 2D PAGE involves gel cutting, and then you have to do gel proteolytic digestion, and extract the peptide, and then do peptide preparation and deposit the peptides onto the MALDI target to do peptide identification. All these procedures are quite time consuming, and also during all these procedures you can dilute and lose your sample along the way.

So what Cepheus does is use a single capillary in touch with the spot of interest, and apply an electric field directly to the spot to electrophoretically extract the protein, and also concentrate the protein right at the tip of the capillary. Once that proceedure is done, we mobilize that protein spot across a miniaturized trypsin membrane reactor. That reactor is directly coupled with the capillary, so the protein is digested there in a matter of seconds, and the other end of the capillary directly deposits the protein of interest on the MALDI target.

We look at Cepheus as the effective technology for coupling 2D PAGE with MS, and then the Gemini is the multidimensional liquid phase and also electrokinetically based technology for large-scale proteome analysis, and then the Orion is the lab-on-chip technology for high throughput and ultrasensitive protein profiling analysis.

What are your long term goals in terms of technology development?

I guess my long term goal is to drive these technologies particularly toward biomarker discovery. Especially during the past six to nine months, our technologies have attracted interest from the medical community. We have developed various collaborations using micro-dissected tissues as one of the approaches for biomarker discovery. The unique feature of micro-dissected tissues is you can separate the cancer cells from the normal tissue cells, so you can really target only protein profiles of cancer tissue cells. They are in tissue, so it's very different from cell culture.

So our objectives are using the unique capabilities of our technologies to study the protein profiles there and discover and develop a panel of biomarkers from there. That would be of great interest from medical diagnosis, to drug discovery, to prognosis of the disease or drug response.

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