1NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Shriners Burn Institute, 3Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, 4Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School
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Righi, V., Apidianakis, Y., Rahme, L. G., Tzika, A. A. Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning. J. Vis. Exp. (38), e1710, doi:10.3791/1710 (2010).
Part 1: Preparing Drosophila for HRMAS Measurements
Part 2: HRMAS Rotor Preparation.
Part 3: HRMAS Data Acquisition
Part 4: Data Processing/Analysis
Part 5: Representative Spectra from Live Drosophila melanogaster Flies
The procedures described above permit to collect reproducible spectra from live Drosophila melanogaster flies. Figures 2 and 3 show representative MR spectra acquired in wild-type (wt) Oregon-R flies. Figure 2 presents 1D 1H HRMAS CPMG spectra. Principal lipid components [CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58ppm), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), CH=CH (5.33 ppm)], glycerol (1,3-CH 4.10 ppm and 4.30 ppm; 2-CH2 5.24 ppm), and small metabolites: β-Alanine (β-Ala, 2.57 ppm), acetate (Ac, 1.97 ppm), phosphocholine (PC, 3.22 ppm) and phophoetanolamine (PE, 3.23ppm) were detected and assigned in accordance with prior reports 4, 5. Signals at 2.02 ppm were assigned to methylene protons of the CH2-CH=CH moiety of mono-unsaturated fatty acids (i.e. palmitoleic). The unsaturated acids were identified by a signal at 5.33 ppm produced by protons of the -CH=CH- moiety. Small metabolites that could not be assigned or were not visible using the 1D spectrum were detected using 2D 1H-1H TOBSY HRMAS (see figure 3).
Figure 1. Experimental set up of in vivo HRMAS 1H MRS for the investigation of live Drosophila at 14.1 T. External standard trimethylsilyl- propionic-2,2,3,3-d4 acid in deuterated water (TSP/D2O). In the square: rotor position at the magic angle in HRMAS probe.
Figure 2. In vivo 1D HRMAS 1H CPMG spectra of a live Drosophila melanogaster wt fly. Lipid components: CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58ppm), Acetate (Ac), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), β-Alanine (β-Ala), phosphocholine (PC) and phophoetanolamine (PE), Glycerol (1,3-CH 4.10, 4.30 ppm; 2-CH2 5.22 ppm), CH=CH (5.33 ppm).
Figure 3. In vivo 2D 1H-1H TOBSY HRMAS spectrum of a live Drosophila melanogaster wt fly at 14.1 T. Small metabolites and lipid components were identified. Metabolites: alanine (Ala), β-Alanine (β-Ala), arginine (Arg), glutamine (Gln), glutamate (Glu), PC phosphocholine (PC), phophoetanolamine (PE), Taurine (Tau), α-Glucose (α-Glc) and Glycerol. Lipids components: CH3 (0.89 ppm), (CH2)n (1.33 ppm), CH2C-CO (1.58 ppm), CH2C=C (2.02 ppm), CH2C=O (2.24 ppm), CH=CH (5.33 ppm).
With the exception of the recent report of the feasibility of in vivo MRI in fruit flies 6, in vivo MRS studies in Drosophila have not yet been reported. In the present protocol, we describe the implementation of a novel in vivo HRMAS 1H-MRS approach for detecting biologically important molecules. Specifically, we detected lipids and small metabolites in live Drosophila flies at 14.1 T in approximately 45 min, which allows adequate acquisition time, while achieving zero fly mortality. The use of a rotor-synchronized WURST-8 adiabatic pulse (C9115) in TOBSY permitted us to obtain a satisfactory SNR and good resolution of tissue spectra relative to use of an isotropic mixing pulse (MLEV-16), in agreement with previous reports 3, 7. Our ability to use TOBSY to detect an improved metabolic profile of Drosophila suggests that TOBSY used with 1D CPMG is well suited for simultaneous qualitative and quantitative analysis of metabolite concentrations and enables improved evaluation of metabolic dysfunction in Drosophila.
Our approach offers biomarkers to investigate biomedical paradigms while advancing the development of novel in vivo non-destructive research approaches in Drosophila, and thus may direct novel therapeutic development.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by Massachusetts General Hospital Institutional Animal Research Review Board Committee.
This work was supported in part by a National Institutes of Health (NIH) grant AI063433 to Laurence G. Rahme, a National Institute Institutes of Health (NIH) Center Grant (P50GM021700) to Ronald G. Tompkins (A. Aria Tzika, Director of the NMR core), and a Shriner's Hospital for Children research grant (#8893) to A. Aria Tzika. We thank Dionyssios Mintzopoulos Ph.D. for assistance in the initial phases of developing this protocol and Ovidiu C. Andronesi Ph.D. for assistance with the TOBSY pulse sequence.
|agar, sucrose, yeast, cornmeal||Food||Genesee Scientific||http://www.flystuff.com/|
|Oregon RS or Canton-S flies||Adult fly lines||Bloomington Stock center||http://flystocks.bio.indiana.edu/|
|2ml tubes||Equipment||Fisher Scientific||K749521-1590|
|Fly incubators||Equipment||high humidity capacity (60-75%), adjustable temperature, and a 12 h:12 h light: dark cycle.|
|Bruker Bio-Spin Avance NMR spectrometer (600.13 MHz) 4mm triple resonance (1H, 13C, 2H) HRMAS probe||Equipment||Bruker Corporation|
|BTO-2000 unit in combination with a MAS pneumatic unit||Equipment||Bruker Corporation|
|4mm zirconium oxide rotor (capacity 50 ul)||Equipment||Bruker Corporation||B3829 (Bruker store)|
|MestReC (Mestrelab Research)||Software||1D NMR spectra analysis
|SPARKY 3, USCF||Software||2D NMR spectraanalysis