Apr 07, 2025

Public workspaceTissue Preparation for Spatial Metabolomics V.2

DOI
https://dx.doi.org/10.17504/protocols.io.d7gf9jtn
  • Guanshi Zhang1,
  • Leila Hejazi1,
  • Dusan Velickovic2,
  • Theodore Alexandrov3,
  • Christopher Anderton2,
  • Kumar Sharma1,
  • Brittney Gorman4
  • 1University of Texas Health San Antonio;
  • 2Pacific Northwest National Laboratory;
  • 3European Molecular Biology Laboratory;
  • 4Pacific Northwest National Lab
Guanshi Zhang
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DOI: https://dx.doi.org/10.17504/protocols.io.d7gf9jtn
Protocol Citation: Guanshi Zhang, Leila Hejazi, Dusan Velickovic, Theodore Alexandrov, Christopher Anderton, Kumar Sharma, Brittney Gorman 2025. Tissue Preparation for Spatial Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.d7gf9jtnVersion created by Guanshi Zhang
Manuscript citation:
Nakayasu et al., 2016. MPLEx: a robust and universal protocol for single-sample integrative proteomic, metabolomic, and lipidomic analyses. mSystems 1(3):e00043-16.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 04, 2025
Last Modified: April 07, 2025
Protocol Integer ID: 126183
Keywords: tissue preparation for spatial metabolomics mass spectrometry imaging, spatial metabolomics mass spectrometry imaging, msi for untargeted metabolomics analysis, mass spectrometry, untargeted metabolomics analysis, metabolomic, assisted laser desorption ionization, detection of metabolite, laser desorption ionization, maldi matrix for positive ion mode analysis, sprayer robotic sprayer for maldi matrix application, analyte extraction from tissue, simultaneous analysis of multiple molecular component, analyte extraction, metabolite, maldi matrix application, laser ionization, sample integrative proteomic, negative ion mode analysis, maldi matrix in order, dihydroxybenzoic acid, integrative proteomic, maldi matrix, lipidomics analysis, multiple molecular component, lipid extraction protocol, maldi sensitivity
Abstract
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).
Guidelines
Key characteristics of TM-Sprayer robotic sprayer for MALDI-specific tissue preparation
1. Patented technology providing very small matrix droplets (<20 microns)
2. High flow rate and fast sample prep (10 to 20 minutes per plate)
3. Highly consistent matrix deposition across entire sample area (+/- 3% by weight)
4. Unique use of temperature and nitrogen flow to control evaporation rate and matrix crystal formation
5. Validated protocols for most matrices (e.g.: DHB, DAN, NEDC)
6. Continuous matrix coverage as needed for high-resolution imaging
7. Rugged operation and easy clean-up

Data types and file formats
Data generated from MS imaging is in the format of either a .d/.mis or .raw (Bruker- and Thermo-based instruments, respectively). These files are converted to the universal MSI file format (.imzML/.ibd) for further data processing and metabolite annotations in SCiLS and METASPACE.

Quality Control
As quality control, in all MALDI imaging optimization, the number of ions annotated by METASPACE (https://metaspace2020.eu/) at 20% false discovery rate (FDR) was used as the main benchmark. All experiments are performed at least in duplicates and results are presented accordingly.

Materials
MATERIALS
ReagentDry ice
ReagentParafilm&trade; M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
ReagentCryoStar NX50 Cryostat with height adjustment, Vacutome, Cold D, 100VThermo FisherCatalog #957090
ReagentCM1950 CryostatLeica Biosystems
ReagentTissue-Tek Accu-Edge Disposable Microtome BladesSakura FinetekCatalog #4689
ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001
ReagentSuperfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
Reagent4 x 6 Zipper Bag
ReagentPocket Scriber Carbide w/Magnet EndCatalog #Westward #4KY32
ReagentFlatbed Scanner
ReagentMTP Slide-Adapter IIBrukerCatalog #8235380
ReagentTissue MALDI TM-SprayerHTX Technologies, LLC
Reagent25-Dihydroxybenzoic acid (DHB >98%)Merck MilliporeSigma (Sigma-Aldrich)
Reagent15-Diaminonaphthalene (DAN 99%)Merck MilliporeSigma (Sigma-Aldrich)
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentWater (miliQ)
ReagentAcetone (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentEthanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentHydrogen chloride solution (HCl 1M)Merck MilliporeSigma (Sigma-Aldrich)
ReagentVacuum Desiccator
ReagentFastPrep-24 HomogenizerMP BiomedicalsCatalog #116004500
ReagentBalance (500 mg)
ReagentContainer to hold dry ice
ReagentWeigh boat
ReagentSpatula
ReagentFalcon 50mL Conical Centrifuge TubesFisher ScientificCatalog #14-432-22
ReagentMetal Bead Lysing MatrixMP BiomedicalsCatalog #6925050
ReagentWild type adult mouse kidneys (avg 200mg/kidney 12 kidneys >2g total)
ReagentSphingomyelin (d18:1/16:0)Cayman Chemical CompanyCatalog #10007946
Reagent13C Sphingomyelin 1:(d18:1/16:0)Cayman Chemical CompanyCatalog #24452
ReagentCentrifuge
ReagentPipette and tips (20 - 300 µL)
Reagent2 mL screw cap vials (1 per layer of the mimetic model)
ReagentWater (deionized)
Reagent3 mL SyringeBecton Dickinson (BD)Catalog #309657
ReagentBeaker
ReagentDry ice-cooled ethanol
ReagentAmmonium Acetate (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentAcetonitrile (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentIsopropyl alcohol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
Reagent2 mL Sorenson MµlTI™ SafeSeal™ Microcentrifuge TubesVWR International (Avantor)Catalog #53550
ReagentWaters autosampler vial
ReagentNEDC (N-(1-naphthyl) ethylenediamine hydrochloride)
STEP MATERIALS
Reagent2 mL Sorenson MµlTI™ SafeSeal™ Microcentrifuge TubesVWR International (Avantor)Catalog #53550
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentTissue MALDI TM-SprayerHTX Technologies, LLC
Reagent4 x 6 Zipper Bag
ReagentMTP Slide-Adapter IIBrukerCatalog #8235380
ReagentFlatbed Scanner
ReagentTissue-Tek Accu-Edge Disposable Microtome BladesSakura FinetekCatalog #4689
ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001
ReagentSuperfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
ReagentPocket Scriber Carbide w/Magnet EndCatalog #Westward #4KY32
ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001
Protocol materials
Reagent4 x 6 Zipper Bag
ReagentMTP Slide-Adapter IIBrukerCatalog #8235380
ReagentFlatbed Scanner
ReagentTissue MALDI TM-SprayerHTX Technologies, LLC
ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001
Reagent2 mL Sorenson MµlTI™ SafeSeal™ Microcentrifuge TubesVWR International (Avantor)Catalog #53550
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)
ReagentTissue-Tek Accu-Edge Disposable Microtome BladesSakura FinetekCatalog #4689
ReagentSuperfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
ReagentPocket Scriber Carbide w/Magnet EndCatalog #Westward #4KY32
Troubleshooting
Snap frozen (liquid N2) sample preparation and sectioning
Remove fresh frozen (liquid nitrogen) kidney sample stored at Temperature-80 °C and place in cryostat set at Temperature-15 °C



Mount the sample on chuck with minimal amount of water (one droplet) and make Thikness7 µm sections, while keeping chuck temperature at Temperature-15 °C and blade temperature at Temperature-20 °C .

ReagentTissue-Tek Accu-Edge Disposable Microtome BladesSakura FinetekCatalog #4689



Thaw-mount in sequential order as outlined in Figure 1. Dry the sections immediately after sectioning in the cryostat chamber. Each slide will be marked with a number via a scribe.


Figure 1. Sandwich model of human kidney biopsy tissue

ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001

ReagentSuperfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15

ReagentPocket Scriber Carbide w/Magnet EndCatalog #Westward #4KY32


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.
ITO slides with mounted coss-sections are then transferred directly from the cryostat to either the vacuum desiccator OR zipper bag.

Reagent4 x 6 Zipper Bag

For imaging later: Place ITO slides in a zipper bag to keep vacuum inside of the bag for storing in the Temperature-80 °C 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.

For immediate imaging: Place ITO slides in the vacuum desiccator at TemperatureRoom temperature for drying Duration00:20:00
Regular glass slides are stored at Temperature-80 °C untiil staining

All slides (regular and ITO) stored at Temperature-80 °C are placed in the vacuum desiccator to defrost

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).
Note
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.

ReagentMTP Slide-Adapter IIBrukerCatalog #8235380

ReagentFlatbed Scanner

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.
Matrix application for MALDI-MSI: Start-up
Turn on TM-Sprayer unit. Set valve to LOAD position.
ReagentTissue MALDI TM-SprayerHTX Technologies, LLC
Launch TM-Sprayer Software
Note
IMPORTANT: Check that exhaust fan is operational. Do not start solvent pump if proper active venting is not functioning.

Start solvent pump at 0.100 mL/min. Backpressure should be normal. ~ 500 psi (3.4 MPa)

Start compressed air flow to TM-Sprayer. Set at 10 psi (70 kPA).
Adjust operating temperature on the sprayer device per solvent mixture.
Note
Follow safety instructions. SOLVENT MIXTURE SHOULD CONTAIN 30% WATER MINIMUM.

Prepare matrix solution. Typical concentration is Concentration5 Mass Percent

Note
Positive ion mode:

DHB is used at Concentration40 Mass Percent in 1:1 MeOH:Water; Temperature80 °C ; 3mm/track spacing; 8 cycles; 1200 mm/min spraying velocity; 0.05 mL/min matrix flow
Reagent25-Dihydroxybenzoic acid (DHB >98%)Merck MilliporeSigma (Sigma-Aldrich)

DAN is used at Concentration4.4 Mass Percent in Amount9 mL of 1:1 Ethanol:Water (Add Amount500 µL of 1M HCl in Amount4 mL milli'Q' water and Amount4.5 mL of ethanol); Temperature90 °C ; 3 mm/track spacing; 16 cycles; 1250 mm/min spraying velocity; 0.05 mL/min matrix flow

Reagent15-Diaminonaphthalene (DAN 99%)Merck MilliporeSigma (Sigma-Aldrich)


Note
Negative ion mode

NEDC is used at Concentration7 Mass Percent in 7:3 Methanol:Water; Temperature70 °C ; 3 mm/track spacing; 8 passes; 1200 mm/min spraying velocity; 0.12 mL/min matrix flow

ReagentNEDC (N-(1-naphthyl) ethylenediamine hydrochloride)


DAN is used at Concentration4.4 Mass Percent in Amount9 mL of 1:1 Ethanol:Water (Add Amount500 µL of 1M HCl in Amount4 mL milli'Q' water and Amount4.5 mL of ethanol); Temperature90 °C ; 3 mm/track spacing; 16 cycles; 1250 mm/min spraying velocity; 0.05 mL/min matrix flow.

Reagent15-Diaminonaphthalene (DAN 99%)Merck MilliporeSigma (Sigma-Aldrich)




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.


With valve in LOAD position, use a syringe to fill loop with matrix.
Note
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.

ReagentConductive ITO Coating Glass Slides for MALDI ImagingBrukerCatalog #8237001

Check that the flow rate of the solvent pump and temperature of the spray nozzle are correct and stable.
Review sample position and define spraying area in TM-Sprayer software.


Select Method by clicking on left column. Current Method field will confirm selection.
Press START. Option changes to CONTINUE. Follow STATUS tab for prompts.
When prompted, switch valve to Spray and confirm by clicking Continue.
Software will automatically delay start to allow purging of liquid lines.
At end of the run the spray nozzle automatically goes to Waste position.
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.
Note
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.

Spray at 0.200 mL/min for Duration00:10:00 , then at 0.010 mL/min until ready to resume.

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.
Set Temperature to Temperature30 °C to start cool down.

Fill syringe with Amount5 mL to Amount6 mL of clean solvent and flush loop completely. Repeat 3 times.

Toggle valve to wash matrix residue. Leave valve in LOAD position.
Keep airflow and solvent pump flow on until temperature is below Temperature50 °C .
Note
This is important to prevent clogging.


Turn N2 flow off (droplet will form at nozzle tip).
Stop solvent pump flow.
Exit TMSP Software.
Power OFF TM-Sprayer. Power OFF solvent pump.
Lipid extraction for LC-MS/MS
Add MilliQ water (Amount200 µL to Amount300 µL ) 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.

Place the sample into a Amount2 mL microcentrifuge tube.
Reagent2 mL Sorenson MµlTI™ SafeSeal™ Microcentrifuge TubesVWR International (Avantor)Catalog #53550

Note
It's been shown these tubes do not leach polymers into the lipid layer from the chloroform.


Add cold (Temperature-20 °C ) chloroform:methanol mix (prepared 2:1 v/v) to sample in 4:1 ratio over sample volume and vortex.
ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)

ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)

Note
i.e. add 400 μl of the 2:1 chloroform:methanol mixture to 80 μl of sample

Vortex for Duration00:00:05 to Duration00:00:10

Let stand on ice for Duration00:05:00

Vortex for Duration00:00:05 to Duration00:00:10
Centrifuge the sample Centrifigation12000 x g, 4°C, 00:10:00 , 5-10 min

Carefully remove the upper aqueous metabolite layer until the interphase contracts without disturbing the protein disk, and discard.
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.
Note
Be sure to gently push out any protein or upper methanol phase that might have entered the pipette tip.

The organic layer (containing lipids) is placed into the speed vac to dry.
Amount500 µL of 2:1 Chl:MeOH and cap (no septa) is stored at Temperature-70 °C until ready for analysis.
Note
Requires extra solution volume for safe storage.

ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)

ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)


Prior to LC analysis, dry sample in speed vac and reconsititute in Amount100 µL of 95:5 MeOH:Chl.

ReagentMethanol (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)

ReagentChloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)