The ambition of the VIB Center for Cancer Biology (CCB) is to contribute to a better understanding of the biology that underlies cancer initiation, progression and metastatic dissemination with the ultimate goal to develop more effective and specific anti-cancer (combination) therapies.
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Metabolic Expertise Center
Managing PI: B. Ghesquière
For optimal functioning and adaptation to different environmental cues, all cells within an organism rely on the fine-tuned equilibrium between numerous metabolites and fluxes of these metabolites through different enzymatic reactions. Consequently, metabolic profiling - more than transcriptomics and proteomics - can provide crucial insights in the actual physiological state of the cell by comprehensive characterization and quantification of small-molecule metabolites.
‘Steady state’-quantification of metabolites of interest (eg TCA cycle intermediates, pentose phosphate pathway intermediates, certain lipids…) under different experimental conditions (pathological vs healthy, knock-out vs wt,…) can result in novel insights and breakthrough findings. However, the real challenge nowadays lies in quantification of fluxes of different metabolites through the complex and intertwined metabolic pathways that are present in a mammalian cell.
13C analysis of metabolites (MFA or metabolic flux analysis) in mammalian cells has become one of the most prominent tools in the understanding and unraveling of metabolic pathways in health and disease. The technology allows a detailed understanding of biochemical activities involved in central metabolism. Starting from 13C –labeled substrates (such as glucose, glutamine, etc), the incorporation of the 13C stable isotope in hundreds of downstream metabolites can be determined, which quantitatively reflects activation or inactivation of specific metabolic pathways. Eventually, the result is a metabolic map representing the distribution of both anabolic as well as catabolic processes in the metabolic network. Based on the comparison of different maps (for instance malignant cells vs control cells) new and important drug targets can be identified.
The VRC masters this innovative technique and is therefore further expanding its existing metabolism research infrastructure with a mass spectrometry-based platform for the 13C analysis of endothelial cells during angiogenesis. This platform is provided with last generation equipment to carry out the separation, quantification, identification and characterization of small molecules. The ultimate goal is to identify the metabolites and corresponding metabolic changes that are triggered during both normal and pathological blood vessel formation and to determine how different tumor cell types adapt their metabolism to gain proliferative advantage over normal cells.
Due to the diversity of the physicochemical properties of the molecules included in the metabolome, it is not possible to study all of them with one single analytical method. To cover a broader range of metabolites, the platform has two different mass spectrometers combined with two different chromatographic methods that enable complementary analyses:
- A triple quadrupole mass spectrometer (7000 GC/MS Triple Quad, Agilent Technologies) linked to a gas chromatography system (GC-MS) to analyze volatile compounds, suitable for small molecule profiling and quantification (absolute or relative).
- A hybrid quadrupole-orbitrap high-resolution mass spectrometer (Q-Exactive, Thermo Scientific) coupled to an ultra-high performance liquid chromatography equipment (UPLC-MS) for polar, non-volatile and high molecular weight molecules, which allows both targeted and untargeted metabolomics approaches.
The platform relies on a scientific-technical team that provides high qualification and expertise in order to advise other scientists in both the experimental design and troubleshooting. They also have an extensive background in the development of new protocols and emerging methodologies and they are currently developing new bioinformatics tools to facilitate the data management and results interpretation.
- GC-MS targeted analysis
- LC-MS targeted analysis
- Quantification of metabolites
- LC-MS untargeted metabolomic profiling
- Isotopomer ratio determination
- Analysis of 13C labeling experiments
Metabolites routinely measured:
Glycolysis: Glucose; Glucose-6-phosphate; Fructose-6-phosphate; Fructose-1,6-bisphosphate; Glyceraldehyde 3 phosphate; 3-phosphoglycerate; 2-phosphoglycerate; Phosphoenolpyruvate; Pyruvate
TCA cycle: Acetyl-CoA; Citrate; Isocitrate; Alpha-ketoglutarate; Succinyl-CoA; Succinate; Fumarate; Malate
PPP intermediates: Glucose 6-phosphate; Ribulose 5-phosphate; Sedoheptulose 7-phosphate; Erythrose 4-phosphate; Glyceraldehyde 3-phosphate; Fructose 6-phosphate
Amino Acids: Alanine; Arginine; Asparagine; Aspartic acid; Cysteine; Glutamine; Glutamic acid; Glycine; Histidine; Isoleucine; Leucine; Lysine; Methionine; Phenylalanine; Proline; Serine; Threonine; Tyrosine; Valine
Urea Cycle: Argininosuccinate; Ornithine; Citrulline
Nucleosides and nucleotides: ADP; AMP; ATP; cAMP; CDP; CMP; CTP; dADP; dAMP; dATP; dCDP; dCMP; dCTP; dGDP; dGMP; dGTP; dTDP; dTMP; dTTP; GDP; GMP; GTP; IMP; UDP; UMP; UTP; Deoxycytidine; Methyldeoxycytidine; Adenosine; Deoxyadenosine; Deoxyguanosine; Thymidine; Formyl-deoxycytidine; Carboxyl-deoxycytidine;
Other molecules: Coenzyme A; Acetyl-CoA; Butyryl-CoA; Malonyl-CoA; Palmitoyl-CoA; Reduced gluthathione; Oxidized gluthathione; NAD; NADH; NADP; NADPH; Creatine