Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Maldi MS spectrometry imaging is an established and useful tool to identify and visualize lipid abundance and distribution, without excessive sample modification. The main advantage of Maldi imaging is its ability to detect the lipids in situ without labeling, and analyze large population of samples simultaneously. Practice makes perfect.

Make sure you practice well before processing the real experimental samples. Minimize the sample preparation time and avoid contamination throughout the procedure. To begin, add the optimal cutting temperature, or OCT into a plastic cryomold to half of the depth of the cryo mold while avoiding bubble formation.

Leave the mold on a flat surface for several minutes and then transfer it onto dry ice. Keep the cryomold flat on the dry ice and allow the OCT to form a flat and even surface. Wait until the OCT is fully solidified in the mold.

Use the frozen OCT stages immediately, or store them at minus 80 degrees Celsius. After anesthetizing the adult flies Using carbon dioxide, prepare a Petri dish containing a piece of laboratory wipe. Use water to moisten part of the wipe to reduce the static electricity.

Keep the wipe half wet and half dry. After cutting the fly head under a dissecting scope one, collect four to five heads and put them onto the dry area of the laboratory wipe. Take the OCT stage from dry ice to microscope two.

Immediately transfer the heads to the OCT stage and arrange them quickly, which takes around 30 to 60 seconds to avoid OCT melting. Leave around one millimeter of blank space around each fly brain to ensure adequate support from the OCT and four to five millimeters of blank space from the edge of the block to provide adequate room to handle the section. Put the OCT stage back onto the dry ice and let it stay on for around three minutes to make sure the OCT stage remains frozen and solid.

After all the fly heads are aligned, let the OCT stage sit on dry ice for another five to 10 minutes. Transfer the OCT stage from the dry ice to a flat surface and then quickly add a large amount of OCT compound to cover all the samples and fill the whole cryomold, which takes around three seconds. Immediately transfer the cryomold back to dry ice and freeze the whole OCT block containing the embedded tissues.

Let the OCT stage sit on the dry ice for another five to 10 minutes. Label the samples on the margin of the cryomold and store the frozen samples at minus 80 degrees Celsius until ready for sectioning. Confirm the ITO coded side by testing the conductivity of the ITO slides using a volt meter set to resistance.

Mark the side with a resistance measurement as the side to adhere the tissue to. Label it and always set a laboratory wipe on the bottom of the slide to avoid slide contamination. Then, allow the tissues to equilibrate in the cryostat chamber for 30 to 45 minutes.

Clean the cryostat, preferably with 70%ethanol. Wipe the roll plate in stage and remove the used blades. Use additional clean wipes to ensure that the ethanol has evaporated and that all the surfaces are dry before sectioning begins.

Next, adjust the temperature of the cryostat chamber sand specimen head according to the type of the tissue. Mount the tissue on the specimen holder using OCT. Be careful to use enough OCT to cover the base of the OCT block and mount the block as flat as possible.

Place a clean blade on the stage and lock it. Position the head of the specimen toward the stage as needed to achieve the desired cutting angle. Then begin the cutting in thick sections until the region of interest is found.

Constantly brush off the extra pieces with a pre-cooled artist brush to keep the stage clean. Adjust the chamber temperature slightly as necessary and change the thickness of the sections to 10 to 12 micrometers once the desired region is reached. Carefully collect the desired section.

Take a room temperature ITO slide and position it over the section. Approach the section gently and adhere it to the slide without leaving traces on the cryostat stage. Place the slide aside in a rack, or laboratory wipe outside the cryostat between the collections of multiple sections.

If comparison across different samples of the same cohort is desired, place the sections from multiple samples onto a single slide so that they can be analyzed simultaneously to minimize variation. If necessary, separate to two slides, as the Maldi target holder can accommodate two slides in a single run. Transport the slides in a vacuum box to a desiccate with desiccate as the bottom layer.

Drive the slides under a vacuum for 30 to 60 minutes, then proceed to the matrix deposition. Use two five dihydroxy benzoic acid, or DHB, in methanol water as the matrix. Place the slides into a slide transporter and securely seal the opening with the wax film.

Seal with the zip bag. Place it into another zip bag containing desiccant. Label the outside and ship the sample to Maldi core facilities with adequate dry eyes.

For the matrix deposition, use 40 milligrams per milliliter of DHB and methanol water as the matrix and spray it using an automatic HTX M5 matrix sprayer. Use nitrogen gas pressure of 10 PSI. Use a Maldi time of flight, MS instrument and positive ion mode to acquire a mass spectrum.

Calibrate the instrument by spotting 0.5 microliters of red phosphorus emulsion in aceto Nitro onto the ITO slides. Use it spectra to calibrate the instrument in the 100 to 1000 MZ mass range by applying a quadratic calibration curve. Set the laser spot diameters to medium as it is the modulated beam profile for 40 micrometer raster width, and gather imaging data by summing 500 shots at a laser repetition rate of 1000 hertz per array position.

Then record the spectral data. Perform the imaging data analysis using root mean square normalization to generate ion images at a bandwidth of plus minus 0.10 Dalton. Align the spectra of both the OCT and brain tissue from the same experiment using the software to evaluate the overlapping peaks in the ion suppression interference of the OCT.

Finally, process the MALDI slides containing the tissue samples by hematoxylin and eosin, or H and E staining. Images from Maldi MS analysis revealed a general decrease in the lipid contents in the LPR1 knockout mutant brain. The representative H and E stained adult fly brain sections are shown here.

The lipid species, their respective MZ values, and the scales of the heat map are also indicated in this image. The average fold of reduction in the LPR1 knockout mutant as compared to the controls from at least four biological replicates, are shown as numbers next to the arrows. The mass spectrum of the selected blank OCT region in the brain tissue region from both the control and mutant flies are shown in the range of MZ1 to 1000 and MZ520 to 900.

The interference of OCT is associated with both ion suppression phenomena and overlapping signal issues. To maintain the fidelity of lipidomics data, appropriate sample preparation, dissection, and cryo sectioning are crucial. After performing the Maldi experiment, the identities of the lipids can be verified through LCMS, also known as liquid chromatography, mass spectrometry based lipodomics.

This technique gains molecular insights on brain lipids homeostasis by using the powerful to soft model system which paves the way to understand human disease related metabolic changes in the brain.