1. Metabolite profiling
A biomarker is any type of biological molecule that can be quantified and that serves as an indicator of physiological status. Within the biomedical research community, there is a great deal of interest in identifying biomarkers for a variety of disease states in an effort to develop more predictive diagnostic procedures. Much of this work focuses on proteins that are involved in cellular signaling pathways. Metabolites can also serve as biomarkers, and my laboratory is developing metabolite profiling strategies for low abundance signaling molecules. The methods that we develop for metabolite separation and quantification are applicable to any complex metabolic network. Our methods also allow us to piece together intricate metabolic networks.

For its relative ease of use in a laboratory setting, our model system is the small flowering plant, Arabidopsis thaliana, and we are characterizing the regulation of the signaling molecule, auxin. Auxin is fundamentally important in plant growth and development as a regulator of numerous biological processes. The primary plant auxin, indole-3-acetic acid (IAA) is tightly regulated through a network of redundant pathways, including biosynthesis, inactivation, transport and signal transduction. We are piecing together these pathways by applying metabolite profiling to Arabidopsis mutants that are defective in some aspect of auxin regulation. In collaboration with a colleague at Boston University, we are examining the role of two specific gene families in IAA biosynthesis; the Cyp79B2 and 3 genes, which convert tryptophan to indole-3-acetaldoxime in the first step of an IAA synthesis pathway, and the tryptophan synthase alpha genes, which may be responsible for the first step in a separate IAA synthesis pathway. Methods include characterization of transgenic knockout lines in Arabidopsis, stable isotope labeling and metabolite profiling with mass spectrometry. Rotation students would participate in weekly group meetings with the Normanly lab and occasional meetings with our BU collaborators.

2. Paclitaxel proteomics project
Paclitaxel (Taxol®) is a natural product (a taxane) of the Taxus plant family that has been shown to be a potent anti-cancer compound. The high demand for this compound in clinical trials and cancer treatment renders harvestation from its natural source (most notably the bark of old growth trees, e.g. the pacific yew) infeasible. Total synthesis is possible, but currently not cost effective. Cultured Taxus cells produce paclitaxel, although the regulatory mechanisms that control paclitaxel accumulation in these cultures are largely unknown. The Normanly, Roberts and Walker labs have an ongoing collaboration to identify the genes responsible for paclitaxel accumulation and to engineer Taxus lines that stably accumulate paclitaxel at high levels. This rotation project approaches the problem of global regulation of paclitaxel accumulation through proteomics. We wish to identify those proteins in Taxus cells that specifically accumulate or diminish during paclitaxel production. Methods employed will be protein extraction, quantification, separation and sequencing. We use two-dimensional gel electrophoresis to separate proteins and mass spectrometry to obtain sequence information. Data obtained from peptide sequencing will be compared to public sequence databases as well as to a sequence database generated by the Walker lab, of Taxus transcripts that are differentially regulated during paciltaxel accumulation. Rotation students will work in the Normanly lab and present their findings in weekly joint group meetings with the Normanly, Roberts and Walker labs.

®Registered Trademark of Bristol-Myers Squibb