Mass Spectometric Identification of Alterations in Metabolic Profiles Due to Genetic Mutations in Plants and Yeast: Abstract No. 254

Introduction: Complete DNA sequences of many organisms are becoming available at rapid pace. However, the functions of most genes thus identified remain unknown. Since metabolites are the end products of most cellular processes, the levels and populations of metabolites in a cell would change due to genetic manipulations. We are developing a rapid method to assign functions to genes by analyzingthe metabolic profiles of wild type and various mutant strains of yeast Saccharomyces cerevisiae and poplar and aspen trees by pyrolysis mass spectrometry and liquid chromatography mass spectrometry (LC-MS) analysis. Methods: Molecular beam mass spectrometry is used in this study to produce mass spectral profiles of the volatile pyrolysis products of the whole cell components from plants andyeast. Direct infusion electrospray mass spectrometry (ESI-MS) and liquid chromatography mass spectrometry are used for the analysis of the cell extracts. Since mass spectra are information rich and very complex, visual identification of spectral changes is difficult. Therefore, multivariate statistical approaches are used to detect and analyze changes in the spectra of compounds resulting fromthe genetic and physiological manipulations. Our research with Saccharomyces cerevisiae involves determining rapid methods to identify changes in metabolic profiles in mutant yeast strains in which the individual open reading frames (genes) have been systematically deleted. Preliminary results: Molecular beam mass spectrometry (MBMS) was used to analyze pyrolysis products of whole cells of wildtype and three strains with different mutations in the aromatic amino acid biosynthetic pathway. Spectra of metabolites with mass range of 20-700 Da were collected and subjected to principal component analysis (PCA). The preliminary results suggest that it should be possible to distinguish the parent strain from the mutants and among different mutants by these methods. Initial investigations ofthe cell extracts are underway using ESI-MS and LC-MS. We have also used the combination of molecular beam mass spectrometry and multivariate statistical analysis to analyze the cell wall composition of genetically-altered poplar and aspen. These studies have allowed us to rapidly identify and assign changes in cell wall composition and modification in the lignin structure resulting from geneticalterations. We will present several examples of using pyrolysis MBMS as a screening method for genetic variation in plants.