Dec 08, 2025
Version 2
MALDI-MSI Lipidomics of Lung Tissue V.2
- 1Pacific Northwest National Laboratory
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Christopher Anderton, Brittney Gorman 2025. MALDI-MSI Lipidomics of Lung Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjdojpvk5/v2Version created by Brittney Gorman
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: December 08, 2025
Last Modified: December 09, 2025
Protocol Integer ID: 234492
Keywords: Multimodal, Serial sections, Lung, hubmap, Cryosectioning, FTICR, matrix, maldi mass spectrometry images of lipid, maldi mass spectrometry image, msi lipidomics of lung tissue, msi lipidomic, mass spectrometry, frozen lung tissue, µm sections of agarose, lung tissue, lipid, high mass resolution fticr, maldi, tissue
Funders Acknowledgements:
Hubmap
Grant ID: U54HL165443
LungMAP HTC URMC
Grant ID: U01HL148861
Abstract
This protocol describes the procedure to obtain MALDI mass spectrometry images of lipids using a high mass resolution FTICR. This protocol is optimized for 12 µm sections of agarose-inflated and carboxymethyl cellulose-embedded fresh-frozen lung tissues.
The embedding method is described in Lukowski et al. doi.org/10.3389/fmolb.2022.1022775 and protocol.io dx.doi.org/10.17504/protocols.io.j8nlkyzx6g5r/v1
Guidelines
Always wear appropriate PPE.
Cryosectioning requires safety glasses, gloves, and a lab coat
All other activities require safety glasses and gloves while handling samples and chemicals
Materials
Instruments:
Bruker Solarix FTICR
HTX M5 Sprayer
Slides:
Fisherbrand‱ Superfrost‱ Plus Microscope Slides
Indium Tin Oxide-coated glass slides (Delta Technologies; Part #: CG-80IN-S115)
Chemicals:
Sodium trifluoroacetate (1 mg/ml)
2,5-dihydroxybenzoic acid
N-(1-naphthyl-) ethylenediamine dihydrochloride
Paraformaldehyde
Ethanol
Methanol
Safety warnings
BSL-2 controls should be used for Human tissue samples
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
Label and number all slides before beginning. Place tissue and slides into the crystat and allow temperature to equilibrate before mounting the tissue (~20 min).
Put up BSL-2 Lab postings on doors
Tissue sectioning
Place sample in cryostat (allow to thermally equilibrate ~20 min)
Set blade temperature -16 °C and specimen temperature of -18 °C
Mount tissue on chunk using a small droplet of water
Cut sections (12 μm) and thaw mount on slides
Mount serial sections on slides:
1. H&E staining (Superfrost plus microscope slide)
2. MALDI (ITO-coated glass slide) - positive ion mode lipidomics
3. MALDI (ITO-coated glass slide) - negative ion mode lipidomics
4. MALDI and Multimodal optical imaging (Superfrost plus microscope slide)
5. MALDI and Multimodal optical imaging (Superfrost plus microscope slide)
Remove slides from cryostat and place in vacuum desiccator (~20 min)
Slide 1 is vacuum-sealed and placed at -80 °C until staining
Section 4x50 μm additional tissue and place in a Sorenson tube for MPLEx (dx.doi.org/10.17504/protocols.io.36wgqd15ovk5/v1)
Autofluorescence Imaging
Autofluorescence Microscopy (AF) is collected on all each tissue section (dx.doi.org/10.17504/protocols.io.q26g75z23lwz/v1)
Matrix Application
Measure out matrix and solvent
Positive ion mode analysis: 2,5-dihydroxybenzoic acid (DHB; 40 mg/mL in 70% MeOH:H2O)
Negative ion mode analysis: N-(1-naphthyl-) ethylenediamine dihydrochloride (NEDC, 7 mg/mL in 70% MeOH:H2O)
Vortex matrix solutions briefly and sonicate for 15 min
On the HTX M5 Sprayer program methods with the following settings:
Positive ion mode (DHB):
Spray temperature: 75 °C
0.05 mL/min flow rate
12 passes
velocity of 1300 mm/s
3 mm offset
40 mm nozzle height
Negative ion mode (NEDC):
Spray temperature 75 °C
0.12 mL/min flow rate
8 passes
Velocity of 1300 mm/s
3 mm offset
40 mm nozzle height
Remove the needle tip and fasten the syringe to the M5 Sprayer line going to the 6-port valve
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.
Make sure that the pump is flowing and pump pressure is 30-40 psi
Place the tissue mounted slides in the M5 Sprayer tray, fasten them with tape.
Move the 6-port valve switch to “Spray”. (Wait for 1-2 min, or until the M5 Sprayer nozzle is spraying the solution)
Once matrix is detected as an opaque solution on a dummy slide, press Start on the M5 Sprayer
When finished, matrix coated slides may be imaged immediately or stored in a desiccator
MALDI imaging MS Acquisition
Put the slide in the MALDI MTP Slide Adapter II and load into Bruker 12T Solarix FTICR
Load the method for Lipid analysis
Note
Parameters of the method:
Step size: 35 μm
Laser focus: Minimum
Ion polarity: Positive
Scan begin: 250 m/z Scan end: 1200 m/z; Time domain 2 M; Resolving power: ~180,000 at 400 m/z; 120 shots with 2000 Hz frequency
Ion polarity: Negative
Scan begin: 200 m/z Scan end: 1200 m/z; Time domain 2 M; Resolving power: ~240,000 at 400 m/z; Ion polarity: positive; 100 shots with 2000 Hz frequency
Calibrate method in ESI mode
Note
- Change from MALDI to ESI
- Turn on the nebulizer
Load syringe with sodium + trifluoroacetate (1 mg/ml) and infuse calibrant
Note
Na-TFA peak list
Calibrate (Enhanced Quadratic)
Change settings back to MALDI and save method
Set up imaging run in Flex imaging
Check signal-to-noise ratio on QC-tissue
Note
Positive peaks:
723.493 m/z; S/N ~1e5
796.53 m/z; S/N ~1e6
835.663 m/z; S/N ~1e6
Negative peaks:
716.524 m/z; S/N ~1e6
766.539 m/z; S/N ~1e7
788.545 m/z; S/N ~1e5
Start Flex imaging run
Matrix Removal
Rinse slides by emerging into fresh 70% methanol for 3 min
Continue on to other imaging modalities
*Optional Before U-FLIP imaging:
- 30 min fixation with 4% paraformaldehyde
- Dehydrated in graded ethanol (75%, 96%, 100%)
- Dried under desiccation and vacuum sealed until imaging