Metabolomics involves the systematic interrogation of the abundance of small chemical molecules (metabolites) within cells, tissues, organs, and organisms. In parallel with high-throughput technologies that facilitate genomic, transcriptomic, and proteomic analyses of cellular and organismal physiology, technologies for metabolite profiling represent an important source of information about the dynamic state of the cell or tissue that is relevant in both health and disease.
LC-MS metabolomics will be the primary focus of the course and will be applied for both targeted and untargeted analysis of endogenous metabolites and in vitro enzyme reactions. We will use approaches for steady state measurement of metabolite levels as well as assessment of metabolite flux. To complement these LC-MS analyses, there will be experiments involving other methodologies. There will be shorter-term activities with other methodologies, including GC-MS, polarimetric and Seahorse measurement of oxygen consumption, FRET sensors and/or MitoTracker measurements, enzymatic techniques for metabolite measurement, and uptake experiments.
The consistent and extended application of LC-MS reflects the emphasis of the course, and the exposure to other methodologies will allow students to appreciate the utility and complementarity of these methods.
Objectives for students:
• Quantitative and qualitative analysis of LC-MS data using currently available tools (vendor software, Rosebrock tools, XCMS online, Agilent Profinder/Genespring)
• Understanding of common interferences and limitations of LC-MS and GC-MS analysis
• Recognize key issues in experimental design and sample preparation for metabolomics
• Awareness of major biochemical pathways active in commonly used cell types
• Familiarity with methods for determining different types of oxygen consumption
Proposed lab exercises:
• Full scan experiment on knockout/drug treatment, students identify significantly changed metabolites and use MS/MS fragmentation and other methods for identification.
• Experimental treatments will be selected in which the discovery of a phenotype is possible with only one of several analytic/separation methodologies.
• Identification of a significantly changed metabolite in blood/plasma/urine and development of a targeted method for its analysis by QQQ, including determination of LOD/LOQ/linearity and other appropriate method validation
• Measurement of metabolite flux by pulse labeling (i.e. kinetic flux profiling)
• Enzyme assay to determine Vmax and Km (measure on MSD?)
• Enzymatic synthesis of a compound (e.g. sedoheptulose bisphosphate or ribose-1-phosphate) and purification by mass-based or HPLC fractionation
• Non LC-MS methodologies:
o Roche kits for lactate and glucose measurement in culture supernatant
o Seahorse experiment, including all drugs for uncoupled, etc.
o FRET experiment (by cytometry and/or microscopy) for NADH levels and proton gradient sensitive Mitotracker staining
o Nutrient uptake by 14C (potentially amino acids?)
o Glycolytic flux in mammalian cells by 3H
Students will receive hands-on training on Agilent QTOF and Thermo Orbitrap, Q-exactive and Vantage triple quadrupole, and Waters SYNAPT G2-S mass spectrometers.
