New frontiers of Metabolomics: from improved qualitative analysis to an accurate global quantification of metabolites in biological samples

Abstract

Technological achievements in the field of bioanalytical chemistry and bioinformatics have allowed for spearheading of metabolomics towards absolute metabolite quantification in biological systems. In the past decade metabolomics community strongly emphasized importance of quantitative data in biological studies as they describe accurately the absolute level of metabolites of interest. Nevertheless, more than 90% of published metabolomics studies still describe relative or semiquantitative data, whilst only 10% of published metabolomics related articles are based on absolute metabolite quantification. Although, this extremely challenging area is heavily relies on bioinformatics tools, current state of metabolomics combined with available genome sequencing data and other system-wide approaches was found to be a powerful hypothesis generating tool which has been widely employed and demonstrated its uniqueness in modern biology. This PhD thesis represents a metabolomics study which includes a method development for absolute non-targeted quantitation of metabolites followed by application of the developed method for quantitative metabolite profiling of Sauvignon blanc grape juice and wine aiming to better understanding Saccharomyces cerevisiae metabolism involved in volatile thiol biosynthesis during wine fermentation. A novel calibration curve-free GC-MS method based on isotope-coded derivatisation for absolute non-targeted quantitation of metabolites accompanied by novel free R-based software MetabQ is presented. MetabQ was created for automated data processing of generated GC-MS data files performing extraction and calculation of absolute metabolite values. Developed method was validated and applied for analysis of more than 250 Sauvignon blanc grape juice and wine from three vintages as part of Sauvignon blanc II Programme. Generated data sets revealed that trace amounts of unsaturated fatty acids in grape juice had strong effect of on yeast metabolism and on development of aroma compounds, and led to a new series of experiments involving shotgun lipidomics and quantitative fatty acid analysis. The lipidomics study demonstrated that juice lipidome consists of less than 15% of free fatty acids that could be acquired by Saccharomyces cerevisiae, and yet, has a strong effect on yeast metabolism. This insight together with finding from Dr. Pinu’s study resulted in set of juice manipulation experiments in order to validate the hypotheses using laboratory-scale wine fermentations.