Professor of Surgical Immunology, Pathology, and Chemistry
University of Michigan
Name: David Lubman
Position: Professor of surgical immunology, University of Michigan, 2005 to present; professor of pathology, University of Michigan 2005 to present; professor of chemistry, University of Michigan, 1991 to present
Background: PhD, chemistry, Stanford University, 1976 to 1979; post-doc, chemistry, Weizmann Institute of Science, 1972 to 1983; research scientist, Quanta-Ray, 1979 to 1983
In 2003, ProteoMonitor spoke with David Lubman about work he was conducting in 2D liquid chromatography and mass spectrometry [See PM 08/08/03]. These days, Lubman has turned his attention to developing a glycoprotein array.
He and his colleagues have done some validation work for the technology, and in an unpublished study have identified two potential cancer biomarkers in pack rats.
They also are using the array on research related to liver, renal, and colon cancer.
ProteoMonitor recently spoke with Lubman about his work. Below is an edited transcript of the conversation.
Give me an update on what you’re working on these days.
There are a number of things we are looking at, but one of the things that we are very interested in is these glycoprotein arrays for biomarker discovery. One of the things we can do is … take serum from disease versus normal, and we can use lectins to pull out the glycoproteins … and spot them on a microarray plate.
We can have a microarray of 150-plus proteins [that] we can pull out of a cancer sample versus, let’s say another slide with 150-plus glycoproteins, which we can pull out of a normal sample, so we can actually make glycoarrays of intact proteins.
And they are fractionated arrays in the sense that we can pull out all the N-lead glycoproteins using these lectins from serum, but then we can use non-porous chromatography to actually separate out those glycoproteins as intact proteins.
In other words … [we] first use lectin affinity chromatography to pull out glycoproteins from serum, and then we can use non-porous chromatography to separate out glycoproteins.
Then we can spot them as basically individual intact glycoproteins on glass slides.
What did you do to make them amenable for analysis of glycoproteins versus proteins?
We can do this technically with proteins too, but in this case we specifically pulled out the N-lead glycoproteins using lectins. We used general lectins like conanavalin-A or one of those lectins to pull out as much of the glycoprotein content as we can and then we separate specifically to glycoproteins.
When we use this we just let all the albumin and everything else go through. We just pull out the proteins we want and make these glass slides and microarrays of glycoproteins.
Then … we can use a number of different lectins, because each of these lectins is specific for a specific glycan structure.
What would someone be able to see with these arrays versus something that’s commercially available?
The commercially available arrays do not have the post-translational modifications. Even if they can somehow make post-translational modifications, they are not like the ones that you find in disease cells. We can make a glycoprotein array of proteins that we have extracted from patient serum, let’s say for cancer.
And then we can make another glycoprotein microarray from normal serum.
Can you use this for other types of PTMs?
We could if there is a way of officially pulling out that PTM.
We could use different lectins against these glycoprotein arrays, and each different lectin is sensitive to a different glycan structure. What happens is you get a very specific pattern of glycan structure for cancer versus normal.
What we do in the second step is once we select our glycoproteins from our cancer array versus the normal and specific lectins that give the pattern that we want, we then go to an antibody array where we take those specific glycoproteins and we place an antibody to the protein on the arrays.
Then … [we] pull out from serum directly that glycoprotein or protein on this antibody array, and we do a sandwich assay where we take the lectin and we see if it lights up the specific structure on that glycoprotein.
We are looking not just at the protein. We are looking at the glycan [that is] on the glycoprotein. Once we do that, these glycans have very specific patterns for disease. We can see the difference between, for example, cancer versus inflammation versus some of the benign conditions versus normals.
Where are you in the development of this? Are you still in the proof-of-principle?
We have actually done some preliminary validation on pack rat cancer. We have not published it yet, it is in review, but we actually have found several potential markers that have very high specificity and sensitivity for pack rat cancer.
How many markers?
We found two that look really good. Now we have only done one lectin against them but at least for initial work, the results are promising. … [We are doing additional work] specifically with different types of lectins because different lectins have a different response and we are actually looking at other cancers too.
We are looking at liver cancer and renal cancer. … We are working on colon cancer very hard right now. The initial results look very interesting. We think we have a couple of potential candidates for colon cancer.
Does the fact that you’re looking at PTMs make what you’re finding more indicative of disease state than a protein that hasn’t been modified?
Yes, what we find is that in the case of proteins, most of the time you are looking for up- and down-regulations of a protein. And we find that if you look at a large number of samples between, let’s say cancer and normal, there is always a population distribution where there is an overlap between the two, and the overlap can be quite extensive.
Whereas we are finding in the case of these PTMs that it is a lot cleaner. There is always a little bit of overlap but it looks like the discrimination is much better.
Arrays have been kind of a secondary technology to mass spectrometry in proteomics. Would you agree with that?
No, not really. We think it is becoming a very important primary technology … especially for certain types of studies that you cannot do by mass spec. For example, antibody type arrays … have become very important. Also certain types of protein arrays, we are doing autoantibody response and we are looking at natural protein arrays to do that.
But there are other important technologies that are coming out … like Joshua LaBaer’s [nucleic acid programmable protein array] technology or various other technologies where people are developing these protein arrays, some of which can produce PTMs and look at things like the autoantibody response. And we are looking at that too, the autoantibody response in cancer patients versus other diseases and normal.
Is the inability of commercially available arrays to recapitulate to specific diseases a bottleneck to the technology?
We are not sure, but we think that if the PTMs for specific disease are important in autoantibody response, that’s going to be an issue.
What are you doing in mass spectrometry?
We used to do technology development but we are not actually building mass specs anymore. Rather what we are doing is mass specs that interface to the array technology, and one of the things that we have been developing is actually being able to look at proteins captured by antibodies on the glass slide using MALDI mass spec.
There was too much activity on the part of instrument companies in terms of development.
Has your MS worked helped you in your array development work?
In fact, one of the mass specs that we invented back in the 1990s was the QIT-TOF, which has been commercialized by Shimadzu, and we have the commercial version of the MALDI QIT-TOF, and we are actually using that to look at the proteins captured on slides.
We can do direct analysis of the glycoprotein spots captured on the antibody arrays using this technology.
How unique is this approach? Most people seem to do only mass spec work or array work. There aren’t very many doing both.
There has been some work [with] people trying to use mass spec to look at microarrays using a variety of techniques but not a lot, obviously. Often the people doing it are not intimately involved in using the arrays to do something. It is more just technology development.
In this case we really needed technology development to further what we are doing in terms of the array work. For example, we can tell whether there is cross reactivity on our antibodies or whether the antibodies are saturated using the mass spec.
Being at the medical school, what sort of interest are you seeing from clinicians in proteomics?
I think there is a great deal of interest in what it can do, especially in terms of … biomarkers and also cell-cell signaling. …In both cases that is due to post-translational modifications and protein expression. You cannot necessarily get that from genomic type studies or RNA type studies. It does not necessarily translate.
What we are finding, especially in biomarker studies, is that post-translation modification is very important, and so there is a lot of interest in proteomics. In addition to cell-cell signaling, looking at phosphorylation cascades and the proteins are actually doing things in the cell. That is where this becomes important.