TITLE: Tissue Preparation for Spatial Metabolomics
AUTHORS: Guanshi Zhang, Dusan Velickovic, Annapurna Pamreddy, Jessica Lukowski, Theodore Alexandrov, Chris Anderton, Kumar Sharma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mass spectrometry imaging (MSI) is a cutting-edge molecular technology that enables simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs. For MSI, cryosections are prepared from flash-frozen tissue and mounted on a contuctive glass slide. We use matrix-assisted laser desorption ionization (MALDI)-MSI, where tissue sections are coated with a MALDI matrix in order to facilitate laser ionization and detection of metabolites with mass spectrometry. We do it using an automated robotic sprayer (TM-Sprayer) with 2,5- dihydroxybenzoic acid (DHB) MALDI matrix for positive ion mode analysis, N-(1-naphthyl) ethylenediamine hydrochloride (NEDC) for negative ion mode analyses, or 1,5-Diaminonaphthalene (DAN) for dual-polarity analysis, prior to being loaded into the MALDI-Q Exactive HF-X Orbitrap-MSI or MALDI-FTICR-MSI for untargeted metabolomics analysis. We have optimized a method for matrix application to maximize analyte extraction from tissue and increase MALDI sensitivity and to create the most homogenous matrix films possible for best lateral resolution of lipids. A key capacity that will be critical for scale-up in KPMP is the use of a TM-sprayer robotic sprayer for MALDI matrix application. This will make inter-lab studies viable, by increasing the reproducibility of sample preparation and is currently in use at UTHSA and PNNL. Our lipid extraction protocol for LC-MS/MS is a robust and universal protocol for single-sample integrative proteomics, metabolomics, and lipidomics analyses (see citation).  </div></div>

[STEPS]
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Remove fresh frozen (liquid nitrogen) kidney sample stored at   and place in cryostat set at
-80 °C
-15 °C
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Mount the sample on chuck with minimal amount of water (one droplet) and make   sections, while keeping chuck temperature at   and blade temperature at  .
-15 °C
-20 °C
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Thaw-mount in sequential order as outlined in Figure 6. Dry the sections immediately after sectioning in the cryostat chamber. Each slide will be marked with a number via a scribe.
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Tissues will be cut to generate adjacent slides that are alternatively subjected to histological staining or MALDI-MSI. This sandwich like assignment allows proper orientation and localization of each MALDI-MSI ion image to its histological counterpart.
?. [Snap frozen (liquid N2) sample preparation and sectioning]
ITO slides with mounted coss-sections are then transferred directly from the cryostat to either the vacuum desiccator OR zipper bag.
?. [Snap frozen (liquid N2) sample preparation and sectioning]
For imaging later: Place ITO slides in a zipper bag to keep vacuum inside of the bag for storing in the   freezer (sectioned samples can be stored in the freezer for months but should be analyzed as soon as possible). When removing the bag from the freezer, keep it close for ~5 minutes to reach RT before opening it.
-80 °C
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Regular glass slides are stored at  untiil staining
-80 °C
?. [Snap frozen (liquid N2) sample preparation and sectioning]
All slides (regular and ITO) stored at   are placed in the vacuum desiccator to defrost
-80 °C
?. [Snap frozen (liquid N2) sample preparation and sectioning]
For the 15T Fourier Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometry, MALDI imaging slides are mounted in the Bruker MTP Slide Adapter II and scanned with a flatbed scanner with at least 3200 dpi resolution (output JPEG, TIFF, or PNG).
This is performed so that the optical image can be co-registered with the imaging experiment run. Visible fiducials (e.g., "X" marks) are previously placed onto MTP Slide Adapter to use as teaching points to register the optical and MS images.
?. [Snap frozen (liquid N2) sample preparation and sectioning]
Visible fiducials (e.g., "X" marks) are previously placed onto MTP Slide Adapter to use as teaching points to register the optical and MS images.
?. [Snap frozen (liquid N2) sample preparation and sectioning]
For immediate imaging: Place ITO slides in the vacuum desiccator at  for drying
0 Room temperature
?. [Matrix application for MALDI-MSI: Start-up]
Turn on TM-Sprayer unit. Set valve to LOAD position.
?. [Matrix application for MALDI-MSI: Start-up]
Launch TM-Sprayer Software
IMPORTANT: Check that exhaust fan is operational. Do not start solvent pump if proper active venting is not functioning.
?. [Matrix application for MALDI-MSI: Start-up]
Start solvent pump at 0.100 mL/min. Backpressure should be normal. ~ 500 psi (3.4 MPa)
?. [Matrix application for MALDI-MSI: Start-up]
Start compressed air flow to TM-Sprayer. Set at 10 psi (70 kPA).
?. [Matrix application for MALDI-MSI: Start-up]
Adjust operating temperature on the sprayer device per solvent mixture.
Follow safety instructions. SOLVENT MIXTURE SHOULD CONTAIN 30% WATER MINIMUM.
?. [Matrix application for MALDI-MSI: Start-up]
Prepare matrix solution. Typical concentration is
Positive ion mode:DHB is used at   in 1:1 MeOH:Water;  ; 3mm/track spacing; 8 cycles; 1200 mm/min spraying velocity; 0.05 mL/min matrix flowDAN is used at   in   of 1:1 Ethanol:Water (Add   of 1M HCl in   milli'Q' water and   of ethanol);  ; 3 mm/track spacing; 16 cycles; 1250 mm/min spraying velocity; 0.05 mL/min matrix flow
Negative ion modeNEDC is used at   in 7:3 Methanol:Water;  ; 3 mm/track spacing; 8 passes; 1200 mm/min spraying velocity; 0.12 mL/min matrix flowDAN is used at   in   of 1:1 Ethanol:Water (Add   of 1M HCl in   milli'Q' water and   of ethanol);  ; 3 mm/track spacing; 16 cycles; 1250 mm/min spraying velocity; 0.05 mL/min matrix flow.NOTE: These are, for now, the optimal condition for matric spraying based on METASPACE output and spatial resolution achieved, and we will continue to optimize both matrix applications.
?. [Matrix application for MALDI-MSI: Start-up]
With valve in LOAD position, use a syringe to fill loop with matrix.
20% overfill is recommended (e.g. Use 6 mL syringe to fill 5 mL loop)
?. [Matrix application for MALDI-MSI: Sample preparation]
Bring ITO glass slide with affixed sample(s) from dessicator.
?. [Matrix application for MALDI-MSI: Sample preparation]
Check that the flow rate of the solvent pump and temperature of the spray nozzle are correct and stable.
?. [Matrix application for MALDI-MSI: Sample preparation]
Review sample position and define spraying area in TM-Sprayer software.
?. [Matrix application for MALDI-MSI: Sample preparation]
Select Method by clicking on left column. Current Method field will confirm selection.
?. [Matrix application for MALDI-MSI: Sample preparation]
Press START. Option changes to CONTINUE. Follow STATUS tab for prompts.
?. [Matrix application for MALDI-MSI: Sample preparation]
When prompted, switch valve to Spray and confirm by clicking Continue.
?. [Matrix application for MALDI-MSI: Sample preparation]
Software will automatically delay start to allow purging of liquid lines.
?. [Matrix application for MALDI-MSI: Sample preparation]
At end of the run the spray nozzle automatically goes to Waste position.
?. [Matrix application for MALDI-MSI: Sample preparation]
Follow end of run prompts. Software will keep track of usage and remaining matrix volume.
?. [Matrix application for MALDI-MSI: Pause Mode]
At the end of sample prep sequence, swith the valve back to LOAD.
IMPORTANT: Keep solvent pump flow on so that clean solvent flows to nozzle and prevents matrix residues from crystallizing and clogging the capillary and spray nozzle.
?. [Matrix application for MALDI-MSI: Pause Mode]
Spray at 0.200 mL/min for  , then at 0.010 mL/min until ready to resume.
?. [Matrix application for MALDI-MSI: Pause Mode]
To resume, start at step 16.
?. [Matrix application for MALDI-MSI: Shut down]
Switch valve back to LOAD position. Set solvent pump flow rate at 0.500 mL/min.
?. [Matrix application for MALDI-MSI: Shut down]
Set Temperature to   to start cool down.
30 °C
?. [Matrix application for MALDI-MSI: Shut down]
Fill syringe with  to   of clean solvent and flush loop completely. Repeat 3 times.
5 ml
6 ml
?. [Matrix application for MALDI-MSI: Shut down]
Toggle valve to wash matrix residue. Leave valve in LOAD position.
?. [Matrix application for MALDI-MSI: Shut down]
Keep airflow and solvent pump flow on until temperature is below .
50 °C
This is important to prevent clogging.
?. [Matrix application for MALDI-MSI: Shut down]
Turn N2 flow off (droplet will form at nozzle tip).
?. [Matrix application for MALDI-MSI: Shut down]
Stop solvent pump flow.
?. [Matrix application for MALDI-MSI: Shut down]
Exit TMSP Software.
?. [Matrix application for MALDI-MSI: Shut down]
Power OFF TM-Sprayer. Power OFF solvent pump.
?. [Lipid extraction for LC-MS/MS]
Add MilliQ water ( to ) to the biopsy tube containing the remaining fresh frozen (liquid N2) biopsy sample (i.e. not sectioned) and lyse the remaining biopsy sample using a tissue lyser. Quantify the amount of tissue remaining by weight.
200 µl
300 µl
?. [Lipid extraction for LC-MS/MS]
Place the sample into a   microcentrifuge tube.
2 ml
It's been shown these tubes do not leach polymers into the lipid layer from the chloroform.
?. [Lipid extraction for LC-MS/MS]
Add cold () chloroform:methanol mix (prepared 2:1 v/v) to sample in 4:1 ratio over sample volume and vortex.
-20 °C
i.e. add 400 μl of the 2:1 chloroform:methanol mixture to 80 μl of sample
?. [Lipid extraction for LC-MS/MS]
Vortex for  to
?. [Lipid extraction for LC-MS/MS]
Let stand on ice for
?. [Lipid extraction for LC-MS/MS]
Vortex for  to
?. [Lipid extraction for LC-MS/MS]
Centrifuge the sample
Centrifuge: 12000 34, 10 min, 4 10
?. [Lipid extraction for LC-MS/MS]
Carefully remove the upper aqueous metabolite layer until the interphase contracts without disturbing the protein disk,  and discard.
?. [Lipid extraction for LC-MS/MS]
Carefully puncture the protein interphase with a pipette tip, remove the organic lipid phase from the bottom of the tube into a conical bottom Waters autosampler vial.
Be sure to gently push out any protein or upper methanol phase that might have entered the pipette tip.
?. [Lipid extraction for LC-MS/MS]
The organic layer (containing lipids) is placed into the speed vac to dry.
?. [Lipid extraction for LC-MS/MS]
of 2:1 Chl:MeOH and cap (no septa) is stored at  until ready for analysis.
500 µl
-70 °C
Requires extra solution volume for safe storage.
?. [Lipid extraction for LC-MS/MS]
Prior to LC analysis, dry sample in speed vac and reconsititute in  of 95:5 MeOH:Chl.
100 µl