24 hours prior to the experiment, a male fly is placed into a two milliliter tube. Upon brief ice cold anesthesia, the fly is pushed into the NMR rotor insert, then placed into the rotor tube. TSP standard solution is added on top of the rotor insert, and then the rotors are closed with a top cap.
The rotor is introduced into the H-R-M-A-S probe and is positioned at the magic angle 54.7 degrees. The spectrometer is set to acquire one and two dimensional experiments. Finally, the Mr.Spectra of specimens are analyzed.
I'm Valeria Rigi from the NMR Surgical Laboratory at the Department of Surgery at Massachusetts General Hospital and Harvard Medical School. Hi, I'm Y AB from the Laboratory of Molecular Surgery in the Department of Surgery at Massachusetts General Hospital and Harvard Medical School. Hello, I'm Lawrence Achman, the director of Molecular Surgical Laboratory and the Department of Surgery at Massachusetts General Hospital and Harvard Medical School.
And I'm Mario Zika, the director of the NMR Surgical Laboratory at Massachusetts Journal Hospital and affiliated with Aula Martin Center at Harvard Medical School. Today we will show you a procedure for the Molecular Characterization Hall of large Melanogaster using medic ango spinning magnetic resonance spectroscopy In our laboratory, we use this procedure to study in vivo Sila metabolites in order to identify disease biomarkers that contribute to novel therapeutic development. So let's get started.
Start preparing by collecting newly eclo flies for three days and then transfer them to fly vials containing fresh fly food. Incubate the collected flies for five days so that they will be five to eight days old just prior to the experiment. We use mail flies only for our experiments.
Prepare experimental two milliliter tubes for the flies by using a flame heated insulin needle to make a hole in the tube cup. Now place single flies into the tubes containing a piece of fly food 24 hours prior to the experiment. Use a high precision balance to weigh the tubes immediately before and after fly insertion and define each fly's weight by subtracting the two values.
A single male fly usually weighs around 0.7 to one milligram with the flies prepared. Let's look at high resolution magic angle spinning or H-R-M-A-S rotor preparation. To begin.
Place a single tube on ice for less than a minute. To anesthetize the fly inside, lay the fly on a layer of aluminum foil placed on ice and use a soft brush to gently push it into the semis spherical hollow space of the NMR insert. Ensure that the fly is completely inserted while avoiding any injuries.
Place the insert into the zirconium oxide rotor tube to locate the flies between the rotor insert and the bottom of the rotor. Now close the insert with a screw and cover it with paraform to prevent any contact between the fly in the TSP standard solution that functions as a reference for both resonance chemical shift and quantification. Add eight microliters of TSB standard solution on top of the rotor insert secure and tighten the whole rotor setup with the top cap with the rotor set up, let's see how to acquire H-R-M-A-S data.
Introduce the rotor into the H-R-M-A-S probe and position it at the magic angle, 54.7 degrees. Next, set the H-R-M-A-S-H one MRS spinning rate to two kilohertz MAS and use an MAS speed controller to stabilize it. Set the temperature to four degrees Celsius with a BTO 2000 unit.
In combination with the MAS pneumatic unit. Flies are kept at four degrees Celsius while in the spectrometer to maintain anesthesia to prepare the magnet, tune and match the coil for optimum performance and shim the magnet for optimal spectra quality. Once shimming is complete, synchronize the INTERPULSE delay to the MAS rotation frequency.
Set the number of transients to 256 with 32, 768 data points. Acquire one dimensional single fly spectrum using a rotor synchronized car, Purcell MEO kill or CPMG spin echo pulse sequence. This sequence works as a T two filter to remove spectral broadening.
Now acquire two dimensional single fly spectrum on all samples using a topsy sequence with atic pulses. Open the rotor and take out the fly. Now let's see how to analyze the data.
We use mare software to analyze the MR Spectra of our specimens. To begin, use a 0.5 hertz line broadening ization function and apply it to all HRM A SH one fis prior to four year transformation. Reference the MR Spectra with respect to TS P at delta equals 0.0 parts per million.
Phase the spectrum manually and apply a Whitaker baseline estimator to subtract the broad components of the baseline prior to peak area calculations. Estimate the peak areas with mastre software. Scale the peak heights with respect to TSP for each acquired spectrum.
Now let's look at some representative MRS results shown here is a representative one DH one H-R-M-A-S-C-P-M-G Spectra acquired in well type Oregon R flies principal lipid components glycerol and small metabolites like beta alanine, acetate, phosphocholine, and phosphoethanolamine were detected and assigned in accordance with prior reports. Signals at 2.02 parts per million were assigned to methylene protons of the propane moiety of monounsaturated fatty acids. The unsaturated acids were identified by a signal at 5.33 parts per million produced by protons of the alkin moiety.
Small metabolites that could not be assigned or were not visible using the 1D spectrum were detected using two DH one H one top Z-H-R-M-A-S. So we have just shown you for the first time how to study the biochemical profile of live drosophila using high resolution magic angle spinning magnetic resonance spectroscopy. When doing this procedure, it's important to use a soft plus to insert the flies without injuring them.
Make sure that you do not tighten the screw too much, thus avoiding breaching the insect and don't acquire spectra for more than an hour because the flies might not survive. So that's it. Thanks for watching and good luck with your experiment.
