NMR Hands On. Nuclear Magnetic Resonance (NMR) Spectroscopy. Small molecules Macromolecules (DNA, RNA, Proteins)
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1 NMR Hands On UAB Metabolomics Training Course July 17-21, 2017 Wimal Pathmasiri and Delisha Stewart NIH Eastern Regional Comprehensive Metabolomics Resource Core (ERCMRC) Department of Nutrition Nutrition Research Institute University of North Carolina at Chapel Hill Nuclear Magnetic Resonance (NMR) Spectroscopy Detects NMR active nuclei Robust and highly reproducible Non-destructive Quantitative Used in Structure elucidation Small molecules Macromolecules (DNA, RNA, Proteins) A number of techniques 1D, 2D, 3D Molecular motion and dynamics Similar method used in Imaging (MRI, fmri) 1
2 NMR Spectroscopy Atom Excitation Emission NMR Frequencies 2
3 NMR Spectroscopy Frequencies in 11.7T magnet Nucleus Basic Frequency (MHz) Natural Abundance (%) 1 H H H C Cl Cl N F P Fe NMR Spectroscopy Explained : Simplified Theory, Applications and Examples for Organic Chemistry and Structural Biology: Neil E Jacobsen, John Wiley & Sons, Inc. 2007, ISBN NMR Spectrometer NMR Console Computer Magnet Pre-amplifier Probe and shim system 3
4 NMR Magnet and the probe Sample is positioned on the probe using a spinner. NMR Experiment 4
5 NMR Spectroscopy Basic 1 H Experiment d1 = delay p1 = pulse width acq = acquisition time 5
6 NMR Signal Fourier Transform Chemical shift (ppm scale) = frequency / Spectrometer Frequency (MHz) Chemical Shift of molecules NMR Spectroscopy Explained : Simplified Theory, Applications and Examples for Organic Chemistry and Structural Biology: Neil E Jacobsen, John Wiley & Sons, Inc. 2007, ISBN
7 Static Magnetic field strengths NMR Spectroscopy Explained : Simplified Theory, Applications and Examples for Organic Chemistry and Structural Biology: Neil E Jacobsen, John Wiley & Sons, Inc. 2007, ISBN Sample Preparation for metabolomics Balance and calibration check Prepare samples on ice, Minimize freeze thaw cycles Dilution Using D 2 O or Buffer (0.2M Phosphate) Extraction MeOH or MeOH/ Water MeOH/ CHCl 3 / H 2 O (Folch Method) 50% Acetonitrile in Water Dry the sample Reconstitute in D 2 O or 0.2M Phosphate Buffer Internal standards Chemical shift reference (DSS, also for line shape) ph reference (Imidazole) Pooled QC Samples Consistency across the whole study is very important 7
8 Sample Preparation for Metabolomics Analysis Current sample preparation practices (in brief) Biofluids Dilute with D 2 O/ buffer/ 0.9% Saline Add internal standard (ISTD, eg. Chenomx) solution or formate (for serum). Centrifuge and transfer an aliquot into NMR tube Tissue and Cells Homogenization performed in ice cold 50/50 acetonitrile/water Supernatant dried down (lyophilized) Reconstituted in D 2 O and ISTD (eg. Chenomx) solution Pooled QC Samples (Sample Unlimited) Mix equal volume of study samples to get pooled QC samples 10% QC samples Pooled QC Samples (Sample Limited) Use independent pool of similar samples 10% QC samples Daily balance and pipette check Samples are randomized for preparation and data acquisition Pooled QC Samples Phenotype 1 Phenotype 2 Equal amounts Aliquots from each sample in the study phenotype are pooled (phenotypic pool) Equal amount of each phenotypic pools are pooled (Combined phenotypic pool) Replicates of pools are prepared Pool samples are prepared along with the study samples Phenotype 1 Phenotype 1 Combined Phenotypes Phenotype 2 t2 Combined Phenotypes Phenotype 2 t1 Pooled samples should cluster tightly 8
9 NMR Data Acquisition 1D NMR 1 st increment of NOESY noesyprid (Bruker) CPMG (serum or plasma) cpmgpr1d (Bruker) To remove broadening of signals due to macromolecules (eg. Proteins and lipids) 2D NMR (for structure elucidation) 2D J-Resolved COSY TOCSY HSQC HMBC Sample Amount in NMR tube Sample volume should cover the NMR Coil Region For optimum field homogenization At least 10% D 2 O in the sample Optimum volume ul (5mm tube) 200 ul (3 mm tube) For very small sample amounts, a NMR with a microcoil probe is an option. Sample gauge is used 9
10 Steps in Data Acquisition Place the sample in the spinner Use sample gauge Tune and match the probe Automatic in new instruments Lock and shim the instrument Gradient shimming Create and set up NMR parameters Acquire data Process the NMR spectrum RTI International ERCMRC Susan Sumner (PI/ Director) Susan McRitchie Program Coordinator Data Analysis Wimal Pathmasiri NMR & GC-MS Tim Fennell Metabolism LC-MS & GC-MS Maria Moreno Biology Kevin Knagge Delisha Stewart Cell Biology & NMR Jessica Gooding LC-MS Yuanyuan Li LC-MS Jason Winnike Keith Levine Metallomics Reza Ghanbari Visiting NIDA Scholar Rose Ewald Intern David Kirchner LC-MS, Biocrates Rod Snyder LC-MS Courtney Whitaker LC/MS Scott Watson Neurotransmitters 10
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