Profiling Methods to Identify Cold-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry

This book chapter describes the analytical procedures required for the profi ling of a metabolite fraction enriched for primary metabolites. The profi ling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profi ling method is adapted to plant material, specifi cally to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and fi nally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verifi cation of cold-regulated metabolites by exact quantifi cation using a GC-MS protocol with extended internal and external standardization.