Metabolomics, Lipidomics and Fluxomics - Mass Spectrometry Lab - NV
Mass Spectrometry based Metabolomics, Lipidomics, and Fluxomics
Mass Spectrometry based Metabolomics, Lipidomics, and Fluxomics
- The Metabolome is the collection of soluble low-molecular weight (50-1500 Da) organic molecules in a biological system.
- Metabolomics is the systematic study of the Metabolome at a given time point of analysis.
- Analysis of the metabolites gives an accurate picture of the physiological status of a cell/ tissue or biofluid, and is important in functional genomics, systems biology, biomarker identification, biomedicine, toxicology etc.
- The two main technologies commonly used in Metabolomics are: Mass spectrometry (MS) and Magnetic Resonance (NMR).
- The NT MS Lab comprises competence and infrastructure for both any type of MS based Metabolomics project, and we have also established method for MS based Lipidomics (i.e. the lipid composition in any sample) and Fluxomics (intracellular carbon flux distribution) research.
Mass spectrometry (MS) is a central technology in Metabolomics due to its very high sensitivity and selectivity. Combined with a separation step before the MS-detection (e.g. gas chromatography (GC), liquid chromatography (LC), convergence chromatography (UPC2), or capillary electrophoresis (CE)) it is a very powerful instrumentation for both quantitative and qualitative analyses of metabolites. There are several different types of mass spectrometers, some better suited for quantitative analyses (single and triple quadrupols) and others for qualitative analyses with requirements of high mass accuracy and high resolution (Time of Flight, Orbitrap, FT-ICR). The physico-chemical properties of metabolites are diverse, ranging from highly charged to hydrophobic species. The consequence is that there is no single GC/LC/UPC2/CE-MS method that completely covers all metabolites in a sample, and a comprehensive analysis of the Metabolome requires the same extract to be analyzed with different methods. Depending on the biological question that is to be answered, different methodological approaches can be used:
- Metabolite Target Analysis: analysis restricted to metabolites of, for example, a particular enzyme system that would be directly affected by a biotic or abiotic perturbation
- Metaboliic profiling: analysis focused on a group of metabolites, for example, a class of compounds such as carbohydrates, amino acids or those associated with a specific pathway
- Metabolic fingerprinting: classification of samples on the basis of either biological relevance and origin
- Metabolic footprinting: a strategy for analysing the properties of cells by looking in a high throughput manner at the metabolites that are excreted or taken up from the surroundings
Metabolomics methods at NT-MS lab
Target quantitative metabolite profiling
At the NT-MS lab we have established six targeted and quantitative Metabolite Profiling methods that cover the major classes of metabolites:
- amino acids,
- organic acids,
- sugar phosphates and other phospho-metabolites,
- nucleotides and nucleosides including pyridine nucleotides (NAD metabolites)
- short and long chain CoA metabolites
Highest level of accuracy and precision is obtained by using internal standard dilution strategy on all these methods (either by commercially available 13C, 15N standards or by using E. coli and yeast 13C- cultivated extracts).
Non-Target Metabolic Profiling
We have established 4 core 10min methods (HILIC and Reverse Phase chromatography with wither positive or negative ESI and MS scan, MS/MS or MSE data acquisition mode on the Synapt HDMS QTof MS) for non-target Metabolomics. Data processing is performed with the Progenesis software form Nonlinear Dynamics/ Waters, and further statistical analysis/ multivariate data analysis is performed with MetaboAnalyst and Unscrambler software. Final stage in the non-target MS Metabolomics work flow is potential identification through data base search (e.g. HMDB, Metlin) and interpretation of the results.
Lipid analysis is of interest in many biological studies, ranging from food applications to lipid status and profiling of healthy vs diseased individuals for a range of health conditions. Analysis of the lipidome by Mass Spectrometry in combination with efficient separation technologies as LC, SFC (supercritical fluid chromatography), GC is extremely promising due to high sensitivity and selectivity.
- We have established a SFC-MS/MS method with base lien separation of all major lipid classes (see reference list Bartosova et al 2021)
Fluxomics is based on cultivation with 13C-labeled substrate, determination of incorporation of 13C-isotope in the different metabolites (i.e. mass isotopomer determination) by MS methodology, a stoichiometric model used for material and isotope balancing, and estimation of intracellular metabolic flux patterns based on the experimental data and the stoichiometric model by using dedicated simulation software.
- We have upgraded our target metabolite profiling method for mass isotopomer determination
- We are using the 13CFLUX2 software for the simulation tasks.
At the Microbial physiology research group we have been working with biological mass spectrometry for over fifteen years (see selected references below). We have focus on both developing new methods incl. sample processing as well as employing these methods on a variety of biological model systems (currently running Metabolome projects on bacteria, yeast, human cells, urine, and cancer tissue).
Professor Per Bruheim
(for complete and updated list check out the Bruheim web pages)
Bartozova, Z., S. Villa Gonzalez, A. Voigt, and P. Bruheim. 2020. High throughput semi-quantitative UHPSFC-MS/MS lipid profiling and lipid class determination. Journal of Chromatographic Science. doi 10.1093/chromsci/bmaa121
Røst, L. M., A. Shafaei, K. Fuchino, and P. Bruheim. 2020. Zwitterionic HILIC tandem mass spectrometry with isotope dilution for rapid, sensitive and robust quantification of pyridine nucleotides in biological extracts. Journal of Chromatography B, Vol 1144, doi 10.1016/j.jchromb.2020.122078
Fuchino, K., U. Kalnenieks, R. Rutkis, M. Grube, and P. Bruheim. 2020. Metabolic profiling of glucose-fed metabolically active resting Zymomonas mobilis strains. Metabolites 10, 81; doi:10.3390/metabo10030081.
Røst, L. M., L. B. Thorfinnsdottir, K. Kumar, K. Fuchino, I. E. Langørgen, Z. Bartosova, K. A. Kristiansen, and P. Bruheim. 2020. Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems. Metabolites 10, 74; doi:10.3390/metabo10020074.
Stafsnes, M. H., L. M. Røst, and P. Bruheim. 2018. Improved phosphometabolome profiling applying isotope dilution strategy and capillary ion chromatography-tandem mass spectrometry. Journal of Chromatography B, 1083: 278-283. doi: 10.1016/j.jchromb.2018.02.004
Kvitvang, H.F.N, and P. Bruheim. 2015. Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry. Journal of Chromatography B, 998:45-9.
Lien, S. K., S. Niedenführ, H. Sletta, K. Nöh, and P. Bruheim. 2015. Fluxome study of Pseudomonas fluorescens reveals major reorganization of carbon flux through central metabolic pathways in response to inactivation of the anti-sigma factor MucA. BMC Systems Biology, 9:6 doi:10.1186/s12918-015-0148-0.
Kvitvang, H. F. K., K. A. Kristiansen, and P. Bruheim. 2014. Assessment of capillary anion exchange ion chromatography tandem mass spectrometry for the quantitative profiling of the phospho-metabolome and organic acids in biological extracts. Journal of Chromatography A, 1370:70-79
Bruheim, P., H.F.N. Kvitvang, and S. Villas-Boas. 2013. Stable Isotop-labeled Derivatization Reagents as Internal Standards in Metabolite Profiling. Journal of Chromatography A, 1296:196-203.
Lien, S.K., H. Sletta, T. E. Ellingsen, S. Valla, E. Correa, R. Goodacre , K. Vernstad, S. E. F. Borgos and P. Bruheim. 2013. Investigating alginate production and carbon utilization in Pseudomonas fluorescens SBW25 using mass spectrometry-based metabolic profiling. Metabolomics, 9: 403-417.
Stafsnes, M. H., M. Dybwad, A. Brunsvik, and P. Bruheim. 2013. Large scale MALDI-TOF MS based taxa identification to identify novel pigment producers in a marine bacterial culture collection. Antonie van Leeuwenhoek, 103: 603-615.
Lien, S. K., H. F. N. Kvitvang, and P. Bruheim. 2012. Utilization of a deuterated derivatization agent to synthesize internal standards for gas chromatography - tandem mass spectrometry quantification of silylated metabolites. Journal of Chromatography A, 1247: 118-124
Kvitvang, H.F., T. Andreassen, T. Adam, S.G. Villas-Bôas, and P. Bruheim. 2011. Highly sensitive GC-MS/MS method for quantitation of amino and non-amino organic acids. Analytical Chemistry. 83: 2705-2711