Gould lab

Physiology and Metabolism Laboratory

: New techniques for metabolic research

Methods for the analysis of metabolites by NMR and mass spectrometry are now becoming well established but technical limitations remain. For example, our understanding of metabolism has been greatly hampered by an inability to image and to measure metabolites in subcellular compartments or even in individual cell-types within complex tissues. In collaboration with Claude Lechene (Harvard Medical School), we have developed new imaging mass spectrometry methods for the quantitation of metabolism with 50 nm spatial resolution. We anticipate many future applications for this powerful new technology and, thus far, have used it to measure the incorporation of dietary and newly synthesized fatty acids into individual lipid droplets of CNS, fat body and midgut cells.

A serious limitation in metabolomics is the difficulty of measuring concentrations of metabolites in sub-microlitre quantities of fluids where the sample volume is not accurately known. To overcome this issue, we developed a technique called volume determination with two standards (VDTS). This approach permits the back calculation of absolute metabolite concentrations from small biological samples of unknown volume. As a proof-of-principle, we applied VDTS to an NMR analysis of polar metabolites from the hemolymph (blood) of tiny Drosophila larvae. In biological contexts where total signal strength differs considerably between samples, we found that VDTS greatly outperforms the widely used analytical method of probabilistic quotient normalization (PQN).

We are greatly assisted in developing new mass spectrometry and NMR methods by James MacRae and Paul Driscoll of the Metabolomics Science and Technology Platform at the Crick.