TITLE:  Spatial N-glycomics with MALDI-MSI for human lung tissue
AUTHORS: Dusan Velickovic, Chris Anderton

[DESCRIPTION]
This protocol describes the procedure to obtain high quality MALDI mass spectrometry images of N-linked glycans from formalin-fixed paraffin embedded tissue. This protocol is optimized for human lung tissue as part of the HubMAP Project. 

[STEPS]
SECTION: Scope
1. This protocol describes the procedure to obtain high quality matrix-assisted laser desorption/ionization (MALDI) mass spectrometry images of N-linked glycans from formalin-fixed paraffin embedded tissue. 
SECTION: Health and Safety
2. Wear nitrile gloves and safety glasses. Follow standard laboratory safety procedures. 
SECTION: Equipment
3. Equipment Required:
SECTION: Equipment
3.1. 8 Coplin jars 
SECTION: Equipment
3.2.   

SECTION: Equipment
3.4. Vegetable steamer (antigen retrieval device)– example: AROMA 8-Cup Cool-Touch Rice Cooker 
SECTION: Equipment
3.5. Flatbed color scanner– example:  EPSON PERFECTION V500 PHOTO)
SECTION: Equipment
3.6. Zeiss PALM MicroBeam
SECTION: Equipment
3.7. Syringe pump capable of 25 µl/min
SECTION: Equipment
3.8. Pump capable of 100 µl/min
SECTION: Equipment
3.9. Automated matrix sprayer – example: TM, M3, or M5-Sprayer from HTX imaging
SECTION: Equipment
3.10. Home-designed chamber for incubation:
Note: This consists of a rubber gasket sealed glass container (example: the jar with lid, KORKEN, IKEA of Sweden. Diameter 11 cm; high 10.5 cm, volume 0.5 L) in which a 50 ml glass beaker and a set of weights is placed.The weights are required to keep the glass beaker from floating.
SECTION: Chemicals &amp; Enzymes
4. Chemicals & Enzymes:
SECTION: Chemicals &amp; Enzymes
4.1. Xylenes
SECTION: Chemicals &amp; Enzymes
4.2. 200 proof ethanol
SECTION: Chemicals &amp; Enzymes
4.3. Water
SECTION: Chemicals &amp; Enzymes
4.4. Citraconic anhydrous buffer
SECTION: Chemicals &amp; Enzymes
4.6. 1 M HCl
SECTION: Chemicals &amp; Enzymes
4.7. PNGase F enzyme (PRIME, 50 U/µg (lyophilized))
SECTION: Procedure
5. Formalin fixed tissue should be sectioned at 5-7 µm and mounted on indium-tin-oxide (ITO) glass slides (25 mm x 75 mm). 
SECTION: Dewax Slides
6.8. Heat slides at 60 °C for one hour.
SECTION: Dewax Slides
6.9. Remove and cool to room temperature, usually 5 minutes. 
SECTION: Dewax Washes
6.11. Xylenes 3 minutes, repeating a total of two times.
SECTION: Dewax Washes
6.12. 100% ethanol 1 minute, repeating a total of two times.
SECTION: Dewax Washes
6.13. 95% ethanol 1 minute
SECTION: Dewax Washes
6.14. 70% ethanol 1 minute
SECTION: Dewax Washes
6.15. Distilled water 3 minutes, repeating a total of two times.
SECTION: Dewax Washes
6.16. Dry slides in desiccator 5 minutes
SECTION: Dewax Washes
6.17. Scan each slide, minus the surrounding sample holder, at a minimum of 1200 ppi resolution using flatbed scanner (as we use for scanning paper document). This will be needed for image registration in FlexImaging during imaging acquisition. Samples for higher resolution will require a higher resolution scanned image. For example, images acquired with a ≤ 50 µm step size require a 2400 dpi scanned image.
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.18. Turn on power supply, and key switch on PALM control unit.
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.19. Mount the slide in the slide holder and place it in the microscope
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.20. Run the PALMRobo software
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.21. In “View” Tab find "Navigation Window".
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.22. In the small screen display at the microscope, select 10x Objective and adjust focusing by turning the knob on the microscope.
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.23. In the "Navigation window" find top left corner of the tissue and select: "Set ROI top left. Next, find bottom right corner of the tissue and select "Set ROI bottom right"
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.24. Click "Scan" 
SECTION: Obtaining  high-resolution (5x-20x) brightfield images using PALM MicroBeam
6.25. Save tile images after scanning is done.
SECTION: Antigen Retrieval Prep
6. Prepare citraconic buffer:
SECTION: Antigen Retrieval Prep
6.1. 25 ml distilled water or HPLC grade water into a 50 ml falcon tube
SECTION: Antigen Retrieval Prep
6.2. Add 25 µl of citraconic buffer to the water
SECTION: Antigen Retrieval Prep
6.3. Add 24 µl of 1 M HCl
SECTION: Antigen Retrieval Prep
6.4. Agitate tube after capping.
SECTION: Antigen Retrieval Prep
6.5. Add water to a total of 50 ml
SECTION: Antigen Retrieval Prep
6.6. Agitate tube to mix
SECTION: Antigen Retrieval Prep
6.7. Check that pH is around 3.0 ± 0.5 by spotting 2 µl of the prepared buffer onto a pH strip
SECTION: Antigen Retrieval Prep
7. Heat slides in vegetable steamer:
SECTION: Antigen Retrieval Prep
7.1. Preheat the vegetable steamer to generate steam by pressing the “cook” switch prior to retrieval procedure (example, preheating takes ~15 min)
SECTION: Antigen Retrieval Prep
7.2. Add ~ 10 ml of the buffer to a 5-slide mailer with side opening
SECTION: Antigen Retrieval Prep
7.3. Place no more than 3 slides per 5-slide mailer with side opening. Slides should be placed with tissue facing outward to the solution in positions 1 and 5, NOT facing the slide mailer walls. Position 3 may face either way
SECTION: Antigen Retrieval Prep
7.4. Completely fill the slide mailer the rest of the way with buffer
SECTION: Antigen Retrieval Prep
7.5. If the mailer has no holes punched in the lid, only snap close one corner of the mailer
SECTION: Antigen Retrieval Prep
7.6. Place the mailer in the corner of the vegetable steamer
SECTION: Antigen Retrieval Prep
7.7. Maintain “cook” option for 30 minutes 
SECTION: Antigen Retrieval Prep
8. Cool the slides after antigen retrieval:
SECTION: Antigen Retrieval Prep
8.1. Remove mailer and place in a tub with cool water from the faucet. Water should not go over the top of the mailer
SECTION: Antigen Retrieval Prep
8.2. Allow to cool for 5 minutes
SECTION: Antigen Retrieval Prep
8.3. Remove half the buffer from the mailer and replace with distilled water.
SECTION: Antigen Retrieval Prep
8.4. Allow to cool 5 minutes on countertop
SECTION: Antigen Retrieval Prep
8.5. Repeat removal of half the buffer two more times, each time with 5 minutes of cooling
SECTION: Antigen Retrieval Prep
8.6. Complete by rinsing in 100% distilled water
SECTION: Antigen Retrieval Prep
8.7. Dry the slides 5 minutes in the desiccator
SECTION: Antigen Retrieval Prep
8.8. Check to ensure scanning of the slides has been performed
SECTION: Antigen Retrieval Prep
8.9. For scanning, scan one slide each at 1200 dpi
SECTION: Application of PNGase F Solution
9. Prepare PNGase F solution: 
SECTION: Application of PNGase F Solution
9.1. Prepare 0.1 µg/µl PNGase F in water: resuspend lyophilized enzyme in 1 mL water 
SECTION: Application of PNGase F Solution
9.2. Ensure that enough solution is prepared, e.g. three full slides takes approximately 1 ml of solution, spraying at 25 µl/min
SECTION: Application of PNGase F Solution
10. Spray the PNGase F solution:
SECTION: Application of PNGase F Solution
10.1. Using the syringe dedicated to PNGase F enzyme solution, rinse the syringe with water by screwing in the needle tip, filling with 3 or more ml of water, and aspirating into waste
SECTION: Application of PNGase F Solution
10.2. Fill with PNGase F solution ensuring that there are no bubbles in syringe. Tip: After loading all the PNGase F solution required, pull a small volume of air into the syringe. Gently dispense the syringe until the large air bubble is gone.
SECTION: Application of PNGase F Solution
10.3. Remove the needle tip and fasten the syringe to the TM-Sprayer line used for PNGaseF. Place the syringe onto the red syringe pump. Check that the syringe head is snug against the dispense head of the syringe pump. Ensure that the diameter is set appropriate to the syringe and the rate is set at 25 µl/min. Do not start the pump at this time. 
SECTION: Application of PNGase F Solution
10.4. Place the samples in the matrix sprayer (e.g., TM-Sprayer) tray, fastening them with tape. 
SECTION: Application of PNGase F Solution
10.5. Set up TM-Sprayer, referring to the guide for the TM-Sprayer. Temperature should be set to 45°C with 15 passes, velocity of 1200, and 3 mm offset. Set temp of the stage to be 40°C
SECTION: Application of PNGase F Solution
10.6. Pressure reading on the front of the TM-Sprayer should be 10 psi.
SECTION: Application of PNGase F Solution
10.7. Start the syringe pump. 
SECTION: Application of PNGase F Solution
10.8. Use a dummy slide to check the TM-Sprayer nozzle for spraying of solution. It generally takes about 1-3 minutes (100 µL) to start spraying.
SECTION: Application of PNGase F Solution
10.9. Once moisture is detected on the dummy slide, press Start on the TM-Sprayer. PNGase F solution will be applied in a thin layer onto target tissue. 
SECTION: Application of PNGase F Solution
11. Incubation PNGase F digest:
To prevent liquid from evaporating too fast and the enzyme from becoming inactive, a wet atmosphere is maintained by placing the ITO slide into a sealed incubation chamber filled with 150 ml saturated KNO3 solution and pre-incubated at 37.5 °C.
SECTION: Application of PNGase F Solution
11.1. After application of PNGase F onto the slide, place it on top of a 50 ml glass beaker in the incubation chamber.
SECTION: Application of PNGase F Solution
11.2. Incubate 2 hours at 37.5 oC
SECTION: Application of PNGase F Solution
11.3. After incubation, remove the slide from the incubation chamber and let dry in the desiccator (15 min). 
SECTION: Application of PNGase F Solution
11.4. Store the slide in a 5-slide mailer to protect the released glycans. If matrix cannot be sprayed the same day, store at -20°C. It is preferred to immediately spray matrix onto the slide. 
SECTION: Application of Matrix
12. Prepare the CHCA Matrix:
SECTION: Application of Matrix
12.1. Prepare CHCA matrix at 7 mg/ml in 50% acetonitrile/0.1% TFA.Add 0.042 g CHCA to 6 ml 50% acetonitrile/0.1% TFA. Prepare fresh each time in a 15 ml falcon tube.
SECTION: Application of Matrix
12.2. Vortex briefly and sonicate 5 minutes.
SECTION: Application of Matrix
12.3. Small chunks may remain in the bottom of the falcon tube. Make sure that there are not loaded into the TM-Sprayer loop as they will clog components of the TM-Sprayer.
SECTION: Application of Matrix
12.4. Filter CHCA solution using Millex (Millipore) 0.2 µm syringe filter.
SECTION: Application of Matrix
13. Spray the CHCA Matrix:
SECTION: Application of Matrix
13.1. Fill the glass-5ml syringe with CHCA solution ensuring that there are no bubbles in syringe. Tip: After loading all the solution required, pull a small volume of air into the syringe. Gently dispense the syringe until the large air bubble is gone. 
SECTION: Application of Matrix
13.2. Remove the needle tip and fasten the syringe to the TM-Sprayer line going to the 6-port valve.
SECTION: Application of Matrix
13.3. Move the switch to “LOAD” and steadily depress the syringe until all the sample is loaded. Note: Do not load air bubbles or undissolved matrix. 
SECTION: Application of Matrix
13.4. Make sure that the pump is flowing at 0.1 ml/minute. Pump pressure should be 30-40 psi when flowing at 0.1 ml/min.
SECTION: Application of Matrix
13.5. Place the sample in the TM-Sprayer tray, fasten them with tape.
SECTION: Application of Matrix
13.6. Set up TM-Sprayer referring to the guide for the TM-Sprayer. Temperature should be set to 80°C with 10 passes, velocity of 1300, and 2.5 mm offset. 
SECTION: Application of Matrix
13.7. Pressure reading on the front of the TM-Sprayer should be 10 psi. 
SECTION: Application of Matrix
13.8. Move the 6-port valve switch to “Spray”.
SECTION: Application of Matrix
13.9. Use a dummy slide to check the TM-Sprayer nozzle for spraying of solution. It generally takes about one minute to start spraying matrix. 
SECTION: Application of Matrix
13.10. Once matrix is detected as an opaque solution on the dummy slide, press Start on the TM-Sprayer. CHCA solution will be applied in a thin layer onto target tissue. 
SECTION: Application of Matrix
13.11. When finished, matrix coated slides may be imaged immediately or stored in a desiccator. 
SECTION: MALDI Imaging MS Acquisition
14. Put the slide in the MALDI holder and load it in the FTICR-MS instrument
SECTION: MALDI Imaging MS Acquisition
15. Load the method for N-glycan analysis Parameters of the method: m/z range 800-4,000; R=512k, laser power=28-32%, laser focus=minimum, laser shots=200, frequency=2,000
SECTION: MALDI Imaging MS Acquisition
16. Teach plate
SECTION: MALDI Imaging MS Acquisition
17. Select measurement region in FlexImaging
SECTION: MALDI Imaging MS Acquisition
18. Run acquisition through FlexImaging
SECTION: MALDI Imaging Analysis
20. Upload imZML files to METASPACE
SECTION: MALDI Imaging Analysis
20.1. Under Annotation settings select: “NGlycDB-v1” for Metabolite database
SECTION: MALDI Imaging Analysis
20.2. Under Annotation settings select: “+Na” as Adducts 
SECTION: Equipment
3.3. pH strips (optimized for acidic pH)
SECTION: Chemicals &amp; Enzymes
4.5. KNO3 
SECTION: Procedure
5.1. Slides should be dried overnight at 37oC (in slide mailer)
SECTION: Dewax Washes
6.10. Use the Coplin jars for dewaxing and washing tissues (by submerging slide mounted tissues) 
SECTION: MALDI Imaging Analysis
19. Open SCiLS Lab
SECTION: MALDI Imaging Analysis
19.1. Load data to SCILS lab
SECTION: MALDI Imaging Analysis
19.2. Convert data to imZML using complete spectra