We present a novel approach to 2D-NMR based metabolomics and demonstrate its utility for the identification of diverse sets of small molecules that differentiate genetically distinct C. elegans strains. We show the value of this method by comparing two
daf-22 alleles to wild type (N2) worms.
daf-22 catalyzes the final step in peroxisomal fatty acid beta-oxidation to produce ascarosides, a class of small molecules that regulate dauer formation as well as several different social behaviors. Many of these ascarosides were identified by comparing 2D-NMR spectra of wild-type and
daf-22 worms using a simple overlay technique (DANS)1. However, DANS was limited to detecting highly conspicuous metabolic changes. Here we report a semi-automated method for comprehensive comparison of metabolomes that combines 2D-NMR with statistical analysis.
To investigate changes in the metabolomes associated with the
daf-22 mutation, we grew liquid cultures of wild type and two different
daf-22 mutant alleles. dqfCOSY spectra, a particularly information-rich form of 2D-NMR spectra, were acquired for both the wild-type and mutant metabolomes. A peak recognition algorithm and principle component analysis (PCA) were utilized to compare the spectra from the three C. elegans strains. Spectral back projections of principle components (PC) were visualized as COSY-like 2D spectra to connect statistical analysis with molecular structure. PCA effectively separated the spectral data sets of the three genotypes; PC1 differentiated both
daf-22 mutant alleles from wild-type C. elegans, whereas PC2 separated the two
daf-22 alleles from each other. Significantly, back projection of PC loadings onto the COSY spectra enabled identification of a large number of metabolites that were up or down regulated in the
daf-22 mutant sets.
The analysis provides insight into the role of peroxisomal beta-oxidation and ascaroside signaling in C. elegans metabolism. We highlight the wide applicability of this metabolomics approach.
(1) Pungaliya C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, Schroeder FC. A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7708-13.