Metabolomics: Methods and Protocols by Oliver Fiehn, Tobias Kind (auth.), Wolfram Weckwerth (eds.)

By Oliver Fiehn, Tobias Kind (auth.), Wolfram Weckwerth (eds.)

Metabolomics: tools and Protocols examines the state of the art in metabolomic research. prime researchers within the box current protocols for the appliance of complementary analytical tools, reminiscent of fuel chromatography-mass spectrometry (GC-MS). Metabolomics: tools and Protocols comprises forward-looking protocols, which supply the fundamental foundation for destiny efforts in elucidating the constitution of the unknowns detected in metabolomic experiences.

Part I of this quantity makes a speciality of GC-MS options; particularly, realization is given to metabolite profiling in blood plasma, building and alertness of mass spectral and retention time index libraries, and metabolomic profiling of common volatiles. partially II, emphasis shifts to information integration and information mining ideas, and particular protocols talk about enhancing trend attractiveness and biomarker choice for platforms point ways, and visualization and research of molecular and metabolomic facts. elements III, IV, and V study the coupling of capillary electrophoresis and mass spectrometry, classical reversed section liquid chromatography-mass spectrometry, and electrochemical detection of metabolites utilizing HPLC separations coupled with coulometric electrode array detectors. partially VI, protocols for the decision of metabolic flux premiums are offered and a theoretical framework for metabolic pathway community research is equipped. the amount concludes with an exam of state of the art NMR protocols.

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Seal GC vials under inert gas using magnetic crimp caps and an adjustable crimpcap sealer. Seal vials in plastic bags with silica gel. Combine the full number of vials comprising one experiment in single bags. 3. Transport sealed bags for short periods at room temperature otherwise on dry ice and store at –20 or –80°C. 4. Allow temperature equilibration at room temperature before opening bags for further analysis. 3. TOF-GC–MS Metabolite Profiling Profiling of metabolite extracts involves a two-step chemical derivatization, which (1) substitutes carbonyl moieties through methoxyamination and (2) comprises a per-silylation prior to the GC–MS analysis of the reaction products (6–9).

6. 7. 8. Erban et al. samples or novel experimental conditions. The following protocol describes a typical analysis that is optimized for 60-mg fresh weight (±5–10%) of dicot leaves. 5). ). The preparation of representative aliquots from large samples, greater than 125 mg (fresh weight), requires homogenization using a precooled mortar and pestle and subsequent generation of small aliquots of the desired amount of material. Keep samples in liquid nitrogen throughout the process. Avoid condensing ice and be careful not to spill the final powder by boiling liquid nitrogen.

4-mL Screw-cap vials (Bester). Polypropylene screw caps with polytetrafluoroethylene (PTFE)-lined rubber septa. 2-mL Wide-mouth crimp-cap vials with crimp caps equipped with polytetrafluoroethylene (PTFE)-lined septa (Bester). 250-µL Vial inserts (Bester). Source of N2 gas. 2. 1. 1. SPME 1. 2. 3. 4. Metal electric grinder, basic analytical mill A11 (IKA, Staufen Germany). Shaking water bath at 37°C. Ultrasonic bath. SPME fiber assembly (65-µm polydimethylsiloxane-divinylbenzene [PDMSDVB]) (Supelco, Zwijndrecht, The Netherlands) (see Note 1).

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