Exo-metabolomics and Succinate Fermentation

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Table of contents

1 Investigation of Acyl-CoA Metabolism in T. brucei
1.1 Methods to Investigate Metabolic Systems in Trypanosoma
1.2 Trypanosomes, Unconventional Organisms
1.3 Metabolomics
1.4 Sample Preparation in Metabolomics
1.4.1 General Considerations
1.4.2 Sampling of T. brucei Metabolome
1.5 Analytical Platforms in Metabolomics
1.6 Isotopes and Isotopic Profiling
1.7 Application of Metabolomics to T. brucei – Selected Illustrations.
1.7.1 Exo-metabolomics and Succinate Fermentation
1.8 Endo-metabolomics, Redox Balances and Gluconeogenesis
1.9 Concluding Remarks
1.10 CoA Thioesters Metabolism in Trypanosomes
1.11 General Considerations
1.12 Acyl-CoA structure
1.13 CoA Biosynthesis
1.14 Functions of CoA and Acyl-CoAs in Metabolism
1.14.1 Acyl Acceptor in Central Metabolism
1.14.2 Fatty Acid and Polyketide Biosynthesis
1.14.3 Degradation of Lipids and Branched Amino Acids
1.14.4 Global Control of Metabolism
1.14.5 Metabolism of Acyl-CoAs in T. brucei
1.15 Objectives of the Thesis
2 Application of Method for Analysis of Acyl-CoA Thioesters
2.1 Introduction to Acyl-CoA Analytical Methodology
2.1.1 Introduction to Acyl-CoA Extraction Methodology
2.2 Application of Separation and Detection of Acyl-CoA thioesters
2.2.1 HPLC Separation of Acyl-CoAs
2.2.2 MS-based Detection of Acyl-CoAs
2.3 Validation of Analytical method
2.3.1 Parameters of Analytical Method
2.3.2 Isotope Dilution Mass Spectrometry (IDMS)
2.3.3 Applied Procedure
2.4 Results of Method Validation
2.4.1 Carry Over
2.4.2 Mass Accuracy
2.4.3 Repeatability and Reproducibility
2.4.4 Calibration Curves and Linearity
2.4.5 Accuracy
2.4.6 Limits of Detection and Quantitation
2.5 Extracting Acyl-CoA thioesters
2.5.1 Acyl-CoA Extraction in E. coli
2.5.2 Acyl-CoA Profile in E. coli
2.6 Analysis of Acyl-CoA Isotopic Profile
2.6.1 Isotopic Profile
2.7 Conclusion
3 Investigation of Acyl-CoA Network in E. coli
3.1 Functional Studies of Metabolic Systems by Instationary 13C-Metabolic Flux Analysis
3.1.1 General Principle of 13C-Metabolic Flux Analysis
3.1.2 Stationary and Instationary 13C-Metabolic Flux Analysis
3.2 Results
3.2.1 Workflow for Functional Analysis of Acyl-CoAs Metabolism in E. coli
3.2.2 Quantitative Metabolomics and Isotopic Analyses of Acyl-CoA Thioesters in E. coli
3.2.3 Labeling Kinetics of Acyl-CoAs and Central Metabolites
3.2.4 Metabolic Model Linking Central Metabolism to Acyl-CoAs Metabolism
3.2.5 Metabolic Fluxes
3.2.6 Propionyl-CoA is Produced by The BCAAs Biosynthetic Pathway
3.2.7 Propionyl-CoA is Produced by The Pyruvate Formate Lyase PflB Under Aerobic Conditions
3.3 Conclusion
4 Acyl-CoA Thioesters in Trypanosoma brucei
4.1 Adaptation of Fast-Filtration method for CoA analysis in T. brucei
4.1.1 Sampling and Quenching
4.1.2 Extraction of Acyl-CoAs
4.2 Development of a New Sampling Method for Metabolomics and Isotopic Profiling Analyses of Acyl-CoA Thioesters
5 Discussion and outlook
5.1 Perspectives from Analytical Point of View
5.2 Perspectives in Metabolic Flux Analysis of E. coli
5.3 Perspectives in Study of T. brucei Metabolism
6 Materials and Methods
6.1 E. coli cultivation
6.1.1 Pre-cultivation E. coli
6.1.2 Cultivation E. coli
6.1.3 Acyl-CoA Quantification in E. coli
6.1.4 Isotopic Profiling
6.1.5 Quantification of Amino Acids and Central Metabolites
6.1.6 Calculation of Metabolite Intracellular Concentration in E. coli
6.2 T. brucei PCF Cultivation
6.2.1 T. brucei sample Collection
6.3 Isotopically Enriched Standards
6.4 IDMS E.coli
6.5 Preparation of Solvents
6.6 HPLC-HRMS Analysis of Acyl-Coenzyme A Thioesters
6.7 HPLC-HRMS Analysis of Amino Acids
6.8 IC-MS analysis of Organic Acids and Phosphorylated Compounds
6.9 Analysis of Extracellular Metabolites
6.10 Data Treatment Software
6.11 Calculation of metabolic fluxes
Bibliography