Monstervoorbereiding voor metabole profilering met behulp van MALDI massaspectrometrie beeldvorming

MALDI Imaging mass spectrometry is a unique advancement in the field of metabolomics that allows us to measure and visualize relative metabolite abundance and distribution, which are indicative of organisms, physiological and pathological conditions. The main advantage of MALDI Imaging is its ability to detect metabolites in C2 without the need of labeling. Demonstrating the procedure, we have you Sami Sauma, a graduate student from Dr.Patricia Casita's lab.

Yuki Chen and Kelly Veerasammy, student researchers from my laboratory. After harvesting, immediately placed the tissue into a liquid nitrogen, cooled, aluminum foil boat and a polystyrene box. Enclose the lid of the polystyrene box to freeze the tissue for 2 to 10 minutes, according to the size of the tissue.

When the tissue is sufficiently frozen, use forceps to remove the boat and transport the tissue secured within the foil on dry ice to the cryostat. Before sectioning the tissue, taking care, not to breathe on the slides. Use gloves and a volt meter set to resistance to test the conductivity of the appropriate number of MALDI compatible indium tin oxide coated glass slides for the analysis.

After labeling, place the slides on a clean paper towel and a 70%ethanol cleaned cryostat set to minus 20 degrees Celsius. Place the tissue sample into the cryostat and set the temperature of the cryostat chamber and specimen head, according to the type of the tissue. After allowing the tissue to equilibrate for 30 minutes, use cryo tissue embedding compound to mount the tissue to the chuck.

And place and lock a clean blade into the stage, Adjust the position of the stage and the angle of the specimen to achieve the desired cutting angle. And section the tissue until the region of interest is revealed. When the desired region has been reached, obtain 10 to 12 micron thick sections, using a pre-cooled brush to carefully, but quickly collect each section onto the labeled side of each slide as they are acquired.

Place a finger under the slides to warm the sections, to ensure secure mounting to the slide. The sections will become transparent in 5 to 10 seconds and turn opaque after about 30 to 60 seconds. When all of the sections have been collected, place the slides in the slide holder and carry on dry ice to a desecrator.

Alternatively, slides can be carried in a vacuum box. Place the slides in a desecrator with desiccant and vacuum dry the slides for 45 to 60 minutes. After drying, if not using immediately, place the slides into the slide transporter and fill the transporter with nitrogen.

Seal the holder with parafilm and place the holder into a zip bag. Then placed the first zip bag into a second labeled zip bag containing desiccant for minus 80 degrees Celsius storage for up to six months. After dehydration, use a bold point silver marker to place X marks on the blank spaces of the slides outside of the tissue sections and use a black fine point marker to place a second black X on top of each silver X.Load one slide into the MALDI slide metal target and place a plastic cover over the slide.

Outline the sample location on the plastic cover and place the slide and MALDI target onto the surface of a flatbed scanner. Then preview the slide and select the desired area. Scan the slide in 16 bit gray scale, in 2, 400 dots per inch and save the image for later use.

To apply matrix to the slides, turn on an automatic matrix sprayer unit, making sure that the valve is positioned at load, and launch the sprayer software. Check that the exhaust fan is operating properly and confirm under the comms tab that the system is communicating correctly. Start the solvent pump at 100 microliters per minute with a back pressure of approximately 500 pounds per square inch.

And set the nitrogen tank to 30 pounds per square inch to start compressed air flow to the matrix sprayer. Adjust the pressure regulator on the front of the sprayer to 10 pounds per square inch, and set the sprayer nozzle temperature as desired. With the valve and the load position, use the syringe to flush the loop with seven milliliters of 70%methanol before filling the loop with six milliliters a matrix.

Place a blank glass slide into the holder and the sprayer, taping down both ends to prevent movement and check that the flow rate and temperature are stable. Press start to set the nozzle temperature and to adjust the pump flow rate, to match the selected method. Switch the valve from load to spray and click continue.

Allowed the system to run to completion. When the deposition is finished, switch the valve from spray to load and click continue. Examine the pattern of matrix coding under a microscope.

If an even layer of fine matrix crystal is observed, deposit the matrix on the appropriate sample slides as just demonstrated. When all of the slides have been treated, clean the system according to the manufacturer's instructions to prevent clogging of the sprayer nozzle. Here, output images from MALDI MS Imaging data analysis of mass discharge spectra selected from every 100 Dalton interval, clearly depicting the utility for the identification of spectra from small molecule metabolites to high molecular weight lipids are shown.

Each road depicts respective, ion heat maps containing both spatial and spectral information of a specific metabolite species across three tissues collected at postnatal days 1, 21 and 60. A strength of the MALDI MSI methodology is the ability to discern the specificity of certain species identified by the mass to charge ratio to developmental milestones or specific anatomical structures. In this analysis, some metabolites were observed to be enriched in postnatal day one neonates or postnatal day 60 adults, or to be uniformly distributed across the tested ages.

Other molecular species were observed to be specifically enriched in gray matter, white matter, or cerebral spinal fluid and ventricles. The spatial distribution of representative metabolites, including hypoxanthine, glutamic acid, N-acetyl aspartic acid, arachidonic acid, and several lipids were also analyzed. To maintain the fidelity of the metabolic stata, appropriate sample dissection, storage and cyro sectioning are crucial to preventing artificial metabolic changes.

After performing your MALDI experiment, you may want to verify the identities of the metabolites you found. This can be achieved by mirco-extraction and liquid chromatography tandem MS.MALDI MS Imaging facilitates metabolomics based inquiry with spatial resolution and affords investigators the opportunity to ascertain metabolic data in a quantitative and visual medium. There's a lot of interest recently to the effect of diet in neurodegenerative disorders, the relationship between the gut microbiome and the brain.

And so there has been a tremendous importance of the study of metabolism in neuroscience, from neuro development to neurodegeneration. The main fact of gut-brain interaction. And it is within this context that the MALDI Imaging facility idea can really provide outstanding technologies to visualize the effect that a specific manipulation might have in the brain.