Metabonomics: Methods and Protocols by Jacob T. Bjerrum

By Jacob T. Bjerrum

This quantity on metabonomics presents particular details at the strategies taken with nuclear magnetic resonance (NMR) spectroscopy, gasoline chromatography-mass spectrometry (GS-MS), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS). Chapters specialise in applied sciences and chemometrics, new release of metabonomics facts, extraction of significant info from information, drug improvement, toxicology, diagnostics, and describing metabonomics as a vital a part of platforms biology. Written within the hugely winning Methods in Molecular Biology series layout, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and fending off identified pitfalls.

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3 citrate ? 2 Chos Malonate? 4 myo ? 1 ? 5 Fig. 3 HSQC NMR spectrum of mouse colon tissue. Key to abbreviation: see caption in Fig. 2; GSH glutathione, tma trimethylamine, hypotau hypotaurine, myo-ino myoinositol, scyllo-ino scyllo-inositol, PCho phosphocholine, GPCho glycerophosphocholines, his histidine 4 Notes 1. Each step of the NMR acquisition can be automated in programs. For each sample a typical NMR acquisition will involve a waiting delay following the introduction of the sample into the magnet to reach constant temperature.

One option is to use Carr-Purcell-Meiboom-Gill (CPMG)-type experiments for the reduction of interfering protein or lipid baseline artifacts. While this approach avoids the detection of the large molecules, NMR signals also produce quantification errors. Another possibility is to physically remove the large molecules using a molecular weight cutoff filter, either 3,000 or 10,000 Da (depending on the size of the polymers). We have been using centrifugal devices (3 and 10 K) to filter blood plasma, human milk, and fecal extracts to remove, respectively, the proteins, large lipids, and polysaccharides.

4) to the cells, and vortex the tube transfer contents to a 2 mL screw cap microcentrifuge tube. (d) Centrifuge tubes again at 800 × g. (e) Remove the buffer and collect in another 2 mL screw cap microcentrifuge tube. (f) Freeze the resulting pellet in liquid nitrogen for 1 min then store at −80 °C. Metabolite Extraction (g) Add 250 µL of methanol: chloroform 2:1 (4 °C) to the frozen pellets. (h) Vortex tubes after 5 min. (i) Dip the tubes into liquid nitrogen for 1 min. (j) Leave the tubes onto ice for 5 min.

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