DESIGN OF METABOLITE RECOVERY PROFILING PROTOCOL

More than 670 GC-MS metabolite profiles were performed in the course of 3 years in an effort to probe robustness and reproducibility of metabolite profiling and to design metabolite recovery as well as the range of metabolite classes which are finally submitted to GC-MS based metabolite profiling. Experiments were performed with two important plant organs, namely root and leaf, using the model plant tobacco, Nicotiana tabacum L. var. Samsun NN (SNN). We investigated solvent composition, pH, and temperature during metabolite extraction and subsequent liquid partitioning of extracts. All permutations of the metabolite profiling protocol were directly compared to the initially published standard protocol. In agreement with the fundamental approach of profiling analyses, results were reported relative to this standard condition. Thus the consistency of results was maintained in the course of years. The resulting set of chromatograms was screened for mass spectral tags (http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/gmd.html). Cluster analyses and multivariate statistical techniques were applied to obtain insight into general trends of protocol variants. A choice of representative metabolites was analysed in depth for potential analytical improvement and final protocol optimization.