Spatial Molecular Imaging of the Glycome Using Mass Spectrometry

We are using exciting, new mass spectrometry-based technologies to map biomolecules in biological samples for spatial biology. Key current challenges include rigor and reproducibility coming from sample preparation and limitations in sensitivity and resolution. To begin, prepare one or more dialysis cups by placing them in high performance liquid chromatography or HPLC water for 15 minutes.

Dialyze 200 microliters of stock isoamylase using a 500 microliter dialysis cup in 14.3 milliliters of HPLC water for two hours at four degrees Celsius without stirring. Using a pipette, gently remove the isoamylase from the dialysis cup and place it into a 1.5 milliliter micro centrifuge tube. Measure the total volume of the isoamylase solution after dialysis.

Based on the stock activity of 200 units per milliliter and the final volume, calculate the total volume required for three units of isoamylase. Pipette aliquots containing three units of isoamylase into thin-walled PCR tubes. Snap freeze the tubes in liquid nitrogen and store them at minus 80 degrees Celsius.

When tissues are processed and ready, prepare the humidity chamber by placing a paper towel soaked with deionized water below the metal rack. Place the chamber in a 37 degrees Celsius incubator for equilibration. Then fill the food steamer reservoir with tap water to the top and set the timer to 60 minutes to turn the unit on.

Place the slide with the tissue facing inward to avoid contact with the sides and to ensure effective antigen retrieval. Fill each slide mailer with the freshly prepared citraconic buffer. Verify that steam is being emitted from the food steamer.

Place the mailers in the steamer and incubate them for 30 minutes. Then transfer the mailers to a deionized water bath for five minutes. Replace half of the citraconic buffer in each mailer with deionized water and allow it to stand for another five minutes.

Remove all solvent and wash the slides by adding 100%deionized water to each mailer, and then removing it. On the back of each slide, draw a circle and a triangle to mark internal standard locations. Apply one microliter of one milligram per milliliter horse radish peroxidase at the circle mark, and one microliter of 10 milligrams per milliliter rabbit liver glycogen at the triangle mark.

Thaw one tube of 100 micrograms of lyophilized peptide N Glycosidase F and one tube containing three units of isoamylase for 10 minutes. Centrifuge the tubes at approximately 2000 G for five seconds. Pipette 50 microliters of deionized water into each lyophilized PNGase F tube.

After vortexing gently, centrifuge the tube at approximately 2000 G for five seconds. Pipette the resulting 50 microliters of PNGase F solution directly into the isoamylase tube and add HPLC water to bring the total volume to one milliliter. Turn on the HTX M5 sprayer and open the HTX M5 software on the connected computer.

Turn off the Knauer pump by pressing the stop button on the front right side and power on the external syringe pump using the switch on the back panel. In the HTX software, under the Methods tab, select the appropriate method for dual enzyme spraying. Navigate to the Temp tab and set the spray nozzle temperature to 45 degrees Celsius.

Open the ultra-high purity nitrogen gas valve and ensure the sprayer pressure is set to 10 pounds per square inch. Using a fresh syringe and needle, fill it with four milliliters of HPLC-grade water. After removing the bubbles, run two milliliters of water through approximately a six inch section of the sprayer line into a waste beaker.

Attach the syringe to the sprayer line and run water through the syringe pump by starting the system at a flow rate of 95 microliters per minute for five minutes. While the syringe pump is flushing, tape the prepared slides to the bottom left corner of the heated tray using a metal alignment guide. Under the Sample tab, define the X and Y parameters for the spray region.

After five minutes, pause the syringe pump and disconnect the syringe. Draw air into it and push the air through the sprayer line to purge any residual water. Then load the prepared dual enzyme solution into a new syringe and connect it securely to the syringe pump.

Attach the loaded syringe containing the dual enzyme solution to the sprayer line. Once the spray nozzle temperature reaches 45 degrees Celsius, press Start under the Cycle tab. When prompted to turn on the Knauer pump, click No.Press start on the syringe pump to begin enzyme spraying.

Place a blank slide under the spray nozzle to confirm visible enzyme deposition. Once the enzyme spray is visibly even, click continue on the software. Next, place the glass cover on the sprayer and monitor for even application, ensuring uniform wetting across the slide surface.

When the spray cycle is complete, press stop on the syringe pump. Turn off the nitrogen gas valve and click Valve Load Confirm in the software. Incubate the sprayed slides in the HTX humidity chamber at 37 degrees Celsius for two hours, ensuring the slides are placed facing upward.

Once incubation is complete, transfer the slides to a desiccator and dry for 15 minutes. Glycogen-derived oligosaccharides and N-linked glycans were detected across a wide master charge range in the liver using matrix-assisted laser desorption ionization mass spectrometry imaging. Glycogen was broadly localized throughout liver hepatocytes while it was absent from vessels and connective tissue in the portal tracts.

The spatial distribution of glycogen and its chain length distribution were both measurable using MALDI MSI. One N-Linked glycan species was widely distributed throughout liver hepatocytes. Another N-linked glycan species displayed a more regional distribution pattern consistent with liver sinusoidal structure.

A distinct and linked glycan localized specifically to portal tract elements. Histological staining confirmed the anatomical structures of the portal tract, including portal vein, bile duct, and connective tissue. We utilize spatial molecular imaging to uncover the cellular, metabolic and molecular underpinnings of cellular biology, physiology, and disease pathology.

These findings will allow molecular imaging of the glycome in diverse biological sources and disease states. Future research will allow detection of different glycome classes and increase the sensitivity and resolution to a single-cell level.