This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.
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…
The authors have nothing to disclose.
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.
Material Name | Tipo | Company | Catalogue Number | Comment |
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Deuterium oxide | Reagent | Sigma-Aldrich | 7789-20-0 | |
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid | Reagent | Sigma-Aldrich | 24493-21-8 | |
agar, sucrose, yeast, cornmeal | Food | Genesee Scientific Corporation | http://www.flystuff.com/ | |
Oregon RS or Canton-S flies | Adult fly lines | Bloomington Stock Center | http://flystocks.bio.indiana.edu/ | |
Paintbrush | Equipment | (size 0) | ||
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 Bio-Spin | ||
BTO-2000 unit in combination with a MAS pneumatic unit | Equipment | Bruker Bio-Spin | ||
4mm zirconium oxide rotor (capacity 50 ul) | Equipment | Bruker Bio-Spin | B3829 (Bruker store) | |
MestReC (Mestrelab Research) | Software | 1D NMR spectra analysis http://mestrelab.com/ |
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SPARKY 3, USCF | Software | 2D NMR spectra
analysis
http://www.cgl.ucsf.edu/home/sparky/ |