Name: Tony Whetton
Position: Head of the School for Cancer and Enabling Sciences at the University of Manchester; Director of the Leukemia Research Fund Mass Spectrometry Unit based at the Wolfson Molecular Imaging Centre, Christie Hospital, Manchester, UK
Background: Professor of Cell Biology, University of Manchester Institute of Science and Technology; Professor of Cancer Cell Biology, University of Manchester
Tony Whetton is the head of the School for Cancer and Enabling Sciences at the University of Manchester, where he has established a mass spectrometry facility for stem cell and leukemia research. His research focuses on downstream proteomic and phosphoproteomic effects of protein tyrosine kinases associated with myeloproliferative disorders and myeloid leukemias.
He's also an adjunct professor in the Department of Gene and Cell Medicine at the Mount Sinai School of Medicine in New York where he uses proteomic approaches in the study of embryonic stem cell differentiation, including the identification of protein markers associated with various stage of differentiation.
Additionally he is a member of an eight-person committee formed at the Human Proteome Organization's annual meeting in 2007 to explore the use of proteomic methods for the evaluation of stem cells in drug development efforts
He spoke recently to ProteoMonitor about his work and the application of proteomics to stem cell research generally. The following is an edited version of the interview.
What led you to begin using proteomics to study stem cells?
My interest was in myeloproliferative disorders and leukemia, which are associated with disregulated oncogenic protein tyrosine kinases. The paradigm example is chronic myeloid leukemia, where you have a t(9;22) translocation which leads to the formation of a chimeric protein called BCR-ABL, and that's a protein tyrosine kinase. With genomic techniques and so forth you're never going to be able to understand the downstream effects of that particular oncogene. Chronic myeloid leukemia was defined many years ago as being a stem cell disease, and therefore there's been a burgeoning requirement for, if you like, cancer stem cell proteomics, since the discovery that tyrosine kinases were oncogenes. And that's where our kind of research started.
Then you come to the related field, which is also active in our lab – embryonic stem cell proteomics. There you find once again that ever since embryonic stem cell technology was developed, there's been a need for understanding how specific signal transduction pathways – the ERK MAP kinase pathway, the GSK pathway – are able to influence the renewal and differentiation of embryonic stem cells. So, there's always been this need for proteomic technology in stem cell biology. I would say it's just that the techniques have gotten better and better to the point where we can now systematically try to understand developmental processes and processes associated with disease within stem cell populations.
So your initial impetus for moving into proteomics was to better understand the role of certain tyrosine kinases in chronic myeloid leukemia?
Yeah, that's one entry point for our group. There were other entry points. If you look at the work we did with Ihor Lemischka at the Mount Sinai School of Medicine in New York, we entered into that collaboration because Igor has an extremely good track record in systematically trying to understand processes of embryonic stem cell self-renewal and differentiation. With Ihor what we wanted to do was to try to systematically understand changes in the proteome associated with embryonic stem cell differentiation to see if post-translational regulation of protein levels was an important level of control for the differentiation and development process.
In the paper that we published in 2009 in Nature we demonstrated that the correlation between changes in chromatic configuration or initiation of RNA synthesis or changes of the level of messenger RNA during a time course of embryonic stem cell differentiation correlated poorly with protein levels in the nucleus. That demonstrates once again the need for proteomics in embryonic stem cell research because essentially we've discovered that microarrays aren't going to give you the whole picture about changes within the phenotype of a developing embryonic stem cell.
Then, if you look at human embryonic stem cells, one of the big issues is, can you develop [protein] markers — cell surface markers preferably — for the differentiation of human embryonic stem cells down specific developmental pathways? In hematology, surface marker antibodies have enabled us to really dissect apart the whole process of hematopoiesis. So surface markers are incredibly useful things. But we don't actually have that many of them available in human embryonic stem cell research. So another driver for studying human stem cell proteomics is to obtain biomarkers for self-renewal and differentiation stages of specific cell populations.
So the aim is to identify proteins that are markers of various stages of differentiation or different directions of differentiation?
Exactly. Identification of a marker on the cell surface enables enrichment of a particular cell population. So the CD34 antigen and antibodies to that antigen have really been of great value to hematology and to peripheral blood stem cell transplantation and so forth. So you want the same kind of markers for other populations because, in the future – and, of course, we're not there yet – in the future we may have cellular therapies that may embryonic stem cell populations or induced pluripotent stem cell populations, and the desire is to be able to enrich for specific subpopulations and thereby specifically select those to take forward for uses in therapeutic strategies.
So if you're able to identify a surface marker for a particular kind of stem cell you'd like to use for therapeutic or other purposes, you could develop immunoassays to enrich for that type of cell?
Yeah, you can pull out those cells because they possess a specific cell surface antigen.
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In a 2006 paper published in Blood you found that roughly 50 percent of differential protein expression in hematopoietic stem cells was due to post-translational regulation. Were you expecting the number to be that high?
I don't think there was a clear knowledge of how protein levels were going to change during differentiation either in primitive hematopoietic cells or embryonic stem cells. So I didn't have any preconceived notions about what the level would be, but 50 percent did surprise me as being a pretty high level because the old DNA-makes-RNA-makes-protein continuum has obviously dominated in molecular cell biology. I would call it quite surprising.
What are the technical challenges involved with proteomic analysis of stem cells?
If you want to look at primary stem cells, as a generality you get very few of them. In the Blood journal study that we published we had very small quantities of primitive hematopoietic cells to work with. Now that we've moved on to chronic myeloid leukemia stem cell populations we're getting even fewer cells back of the true stem cell kind because they are a rare population of cells. So the technical challenge is identifying very low quantities of protein. And that, over the last few years, has become less of a challenge because of the increased sensitivity of mass spectrometers and improved liquid chromatography peptide separation techniques. So we can make pretty serious inroads now into low-abundance proteins within a relatively rare population of cells such as a chronic myeloid leukemia stem cell population.
What kind of mass spec equipment do you use in your work?
We use a variety of different mass spectrometers. We use an AB Sciex [QSTAR] Elite quadrupole/time-of-flight, a 5800 MALDI TOF/TOF from AB Sciex, and a Thermo Fisher Orbitrap Velos instrument. When we want to validate our observations we develop multiple-reaction monitoring [assays] using an AB Sciex 4000 QTRAP.
In 2007 the Human Proteome Organization launched its Proteome Biology of Stem Cells initiative. What are the goals of that program?
We're trying to bring a coherence towards stem cell research to identify specific aims and objectives and unite various labs in best practice. One of our objectives is also to have a paradigm project, so to speak, so that we're able to develop a fuller understanding of the stem cell proteome as a community. There are a series of objectives, like mapping post-translational modifications on transcription factors that govern self-renewal; absolute quantification of key proteins…
Have any types of post-translational modifications emerged as particularly significant with regard to stem cells?
In embryonic stem cells and other stem cell populations, protein phosphorylation remains the major area of interest for most groups in the field.
Do the current restrictions on embryonic stem cell research in the US have any effect on your work?
It has not affected my research to any great extent. I think the phenomenon of induced pluripotent stem cells has offered other avenues for exploration and this is an extremely exciting area, this reprogramming phenomenon. So, if anything, I think more attention should be given towards the induced pluripotent stem cell phenomenon with respect to investigations using techniques such as proteomics, and there are groups that are beginning to address induced pluripotent stem cell proteomics.
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