Quantitative Metabolomics by LC–MS
SUPERVISOR: Gunda KÖLLENSPERGER
Project assigned to: Karin ORTMAYR
A major direction within metabolomics, the youngest "omics" platform, is the quantitative metabolic profiling of the intracellular primary carbon metabolism. Analytical developments focus on the elaboration of suitable sampling strategies, sample preparations and comprehensive MS-based assays for these metabolites in different organisms.
As a matter of fact, up to date, quantifying redox–sensitive metabolites such as NADPH and its redox partner NADP+, the redox couple NAD+/NADH or the components of the intracellular sulfur metabolism remains a highly challenging analytical task. The instability of these compounds is a serious obstacle to their absolute quantification in cell extracts and demands for elaborate evaluation and optimization of the sample preparation process.
One key to success in this endeavor is the production of isotopically labeled standards enabeling isotope dilution strategies. Moreover, these labeled metabolites proved to be extremely valuable tracers for the evaluation of sample preparation. As a drawback for the metabolites addressed in this study, the availability of such standards is limited. Therefore, part of the work will be devoted to the production of isotopically labeled standards by chemical (deuteration) or by in vivo synthesis (13C and 34S labeling of yeast).
Aims and methods.
The aim of this project is the development of analytical methods and workflows for the absolute quantitative analysis of redox-sensitive intracellular compounds of the primary metabolome as well as intermediates of the sulfur cycle. Employed analytical techniques will comprise state of the art mass spectrometric instrumentation used in the field of metabolomics (LC-MS-MS and GC-MS-MS). Moreover complementary LC-ICP-MS (inorganic mass spectrometry) will be introduced for the determination of sulfur and phosphorus containing metabolites. A thorough evaluation and validation of the sample preparation process employing isotopically labeled internal standards will be included.
J. Hou, N. F. Lages, M. Oldiges and G. N. Vemuri (2009) Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae. Metabolic Engineering 11, 253–261
B. Luo, K. Groenke, R. Takors, C. Wandrey and M. Oldiges (2007) Simultaneous Determination of Multiple Intracellular Metabolites in Glycolysis, Pentose Phosphate Pathway and Tricarboxylic Acid Cycle by Liquid Chromatography-Mass Spectrometry. Journal of Chromatography A 1147, 156-164
S. Neubauer, C. Haberhauer-Troyer, K. Klavins, H. Russmayer, M. G. Steiger, B. Gasser, M. Sauer, D. Mattanovich, S. Hann and G. Koellensperger (2012) U13C Cell Extract of Pichia pastoris - A Powerful Tool for Evaluation of Sample Preparation in Metabolomics. Journal of Separation Science 0, 1-15
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