Dec 10, 2024

Public workspaceGel-Assisted Mass Spectrometry Imaging (GAMSI) Protocol 

DOI
dx.doi.org/10.17504/protocols.io.ewov1d4jyvr2/v1
  • 1Department of Chemistry, University of Illinois Chicago;
  • 2Department of Biological Sciences, University of Illinois Chicago
Yat Ho Chan
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.ewov1d4jyvr2/v1
External link: http://www.gaogroup.site/resources.html
Protocol Citation: Yat Ho Chan, Maddison Hibbard, Ruixuan Gao 2024. Gel-Assisted Mass Spectrometry Imaging (GAMSI) Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d4jyvr2/v1
Manuscript citation:
Chan, Y.H., Pathmasiri, K.C., Pierre-Jacques, D. et al. Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics. Nat. Commun. 15, 5036 (2024). https://doi.org/10.1038/s41467-024-49384-w
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: November 19, 2024
Last Modified: December 10, 2024
Protocol Integer ID: 112321
Keywords: Mass spectrometry imaging, Imaging mass spectrometry, Spatial lipidomics, Spatial omics, Spatial metabolomics, Single-cell mass spectrometry imaging, Subcellular mass spectrometry imaging, Single-cell spatial lipidomics, Subcellular spatial lipidomics, Expansion microscopy
Funders Acknowledgements:
NIH
Grant ID: DP2MH136390
Abstract
Gel-Assisted Mass Spectrometry Imaging (GAMSI) is an accessible and robust sample preparation and imaging workflow to enhance the spatial resolution of MALDI-MSI to the single-cell scale and beyond. Using mouse brain sections as an example, we describe a step-by-step GAMSI protocol that increases the spatial resolution of MALDI-MSI ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. The protocol is expected to be applicable to a diverse array of tissue types.
Attachments
Icon for Materials_for_GAMSI.xlsx
Materials_for_GAMSI....
10KB
Image Attribution
Single-cell lipidomic profiling in mouse cerebellum with Gel-Assisted Mass Spectrometry Imaging (GAMSI) (image credit: Gao Lab, University of Illinois Chicago)
Materials
Tissue preparation
MaterialsShape/sizeVendorProduct Number
Isopentane (2-methylbutane)1 LSigma320404-1L
Glass microscope slides25 x 75 mmMidsciMID7100-45

Day 1: Sample fixation
MaterialsShape/sizeVendorProduct number
Paraformaldehyde, 20%10 mLThermo047340.9M
10x PBS500 mLThermo70-011-069
Day 1: Sample anchoring
MaterialsShape/sizeVendorProduct number
Acryloyl-X5 mgThermoA20770
Anhydrous DMSO3 mLThermoD12345
Day 2: Sample gelation
MaterialsShape/sizeVendorProduct number
Sodium acrylate25 gThermo50-750-9773
Acrylamide100 gSigmaA9099
N,N-Methylenebisacrylamide5 gSigma294381
Sodium chloride500 gSigmaS9625
4-Hydroxy-TEMPO (4HT)1 gSigma176141-1G
NNN′N′-Tetramethylethylenediamine (TEMED)25 mLSigmaT7024-25ML
Ammonium persulfate (APS)25 gThermoPI17874
0.5-mil Polyimide (Kapton) tape7.94 mm W x 33 m LCaplinqPIT0.5S-UT/7.94
Day 2: Sample homogenization
MaterialsShape/sizeVendorProduct number
Trypsin50 mgThermo20233
Proteinase K2 mLNEBP8107S
Day 4: Sample expansion and immobilization
MaterialsShape/sizeVendorProduct number
No. 1.5 cover glass36 x 60 mmTedPella260461-100
ITO-coated glass slides70 - 100 Ω/sqDelta TechnologiesCB-90IN-S111,
(Optional) Matrix sublimation and recrystallization
MaterialsShape/sizeVendorProduct number
1,5-diaminonaphthalene (DAN)36 x 60 mmTedPella260461-100
Acetone1 LThermoA929-1
Isopropyl alcohol 1 LSigma1027811000
PYREX petri dishes100 mm diameterThermo08-747C
Recipes and Reagents:

4% PFA
MaterialsAmount
Paraformaldehyde, 20%10 mL
10x PBS5 mL
Water35 mL

Note
4% PFA solution can be stored at Temperature4 °C up to 2 months.

3% PFA/0.1% GA
MaterialsAmount
Paraformaldehyde, 20%7.5 mL
Glutaraldehyde, 8%625 µL
10x PBS5 mL
Water36.875 mL
Note
3% PFA/0.1% GA solution can be stored at Temperature4 °C up to 1 month. Store in Amount1 mL aliquot at Temperature-20 °C for long term storage.


AcX stock (Concentration10 mg/mL )
  • Thaw Acryloyl-X (Amount5 mg tube) to room temperature.
  • Dissolve Acryloyl-X (Amount5 mg tube) in Amount500 µL anhydrous DMSO.
  • Aliquot in Amount20 µL batches.
  • Store in a desiccated environment at Temperature-20 °C .
  • DO NOT reuse AcX after thawing.

4HT stock (Concentration5 mg/mL )
  • Dissolve Amount0.5 g of 4HT in Amount100 mL of water.

TEMED stock (Concentration100 mg/mL )
  • Dissolve Amount10 g of TEMED in Amount100 mL of water.

APS stock (Concentration100 mg/mL )
  • Dissolve Amount10 g of APS in Amount100 mL of water.

Note
Store 4HT, TEMED and APS stock solutions in Amount100 µL aliquots at Temperature-20 °C


Stock-X solution (for 4-fold expansion) (Excluding 4HT, TEMED, APS):
ABCD
Sodium acrylate382.258.55
Acrylamide500.52.5
N,N-Methylenebisacrylamide20.750.15
Sodium chloride29.2411.7
10x PBS10x 11x
Water0.9
Total9.4
A: Materials; B: Stock solution concentration (g/100 mL); C: Amount of stock solution (mL) to be mixed; D: Final concentration (g/100 mL).

Note
  • The final concentration is calculated by accounting for the volumes of 4HT, TEMED, and APS to be added.
  • Follow columns A-C of the table to prepare the Stock-X solution.
  • Store Stock-X in Amount1 mL aliquots at Temperature-20 °C .


Stock-S solution (for 6-fold expansion) (Excluding 4HT, TEMED, APS):
ABCD
Sodium acrylate382.72110.34
Acrylamide502.84014.2
N,N-Methylenebisacrylamide20.0250.005
Sodium chloride29.238.76
10x PBS10x 11x
Water0.084
Total9.67
A: Materials; B: Stock solution concentration (g/100 mL); C: Amount of stock solution (mL) to be mixed; D: Final concentration (g/100 mL).

Note
  • The final concentration is calculated by accounting for the volumes of 4HT, TEMED, and APS to be added.
  • Follow columns A-C of the table to prepare the Stock-S solution.
  • Store Stock-S in Amount1 mL aliquots at Temperature-20 °C .

Trypsin stock (Concentration2.5 Mass / % volume or Concentration25 mg/mL )
  • Dissolve Amount50 mg of Trypsin in Amount2 mL of 1x PBS.
  • Store trypsin stock in Amount100 µL atTemperature-20 °C .















Tissue preparation
Tissue preparation
Euthanize mouse via CO2 asphyxiation followed by decapitation.
Dissect mouse brains and immediately freeze on prechilled isopentane (Temperature-80 °C ).
Note
The dissected mouse brains can be stored at Temperature-80 °C up to 3 months until sectioning.


Temperature
Make a Thikness25 µm cryosection of the dissected mouse brain using a cryostat of choice (e.g., CryoStar NX50 Cryostat, Epredia).
Temperature
Multiple sections can be mounted on a single slide, but they must be gelled together. Thaw-mount the Thikness25 µm brain sections on positively charged glass microscope slides (e.g., MID7100-45, Midsci).
Note
1. Multiple sections can be mounted on a single slide, but they may need to be gelled together in the subsequent steps.
2. The mouse brain sections can be stored at Temperature-80 °C up to 3 months until further analysis and procedure.


Day 1: Sample fixation
Day 1: Sample fixation
30m
30m
Thaw the Thikness25 µm mouse brain sections to TemperatureRoom temperature .
5m
Temperature
Fix the Thikness25 µm mouse brain sections in 4% PFA or a combination of 3% PFA and 0.1% glutaraldehyde at TemperatureRoom temperature for Duration00:10:00 . 
Note
Each mouse brains section on the glass slide requires at least Amount1 mL of fixatives.



10m
Incubation
Pipetting
Remove the fixatives and wash sample 3 x 5 mins in Amount1 mL wwith Amount1 mL Amount1 mL of 1x PBS.
Pipetting
Wash
Remove the fixatives and wash sample Duration00:05:00 in Amount1 mL of 1x PBS. (1/3) 
5m
Remove the fixatives and wash sample Duration00:05:00 in Amount1 mL of 1x PBS. (2/3) 
5m
Remove the fixatives and wash sample Duration00:05:00 in Amount1 mL of 1x PBS. (3/3) 
5m
Day 1: Anchoring
Day 1: Anchoring
6h 10m
6h 10m
Thaw Acryloyl-X (AcX) stock solution (Concentration10 mg/mL ).
5m
Temperature
Dilute the AcX stock solution 1:100 in 1x PBS to a concentration of Concentration0.1 mg/mL . Vortex well.
5m
Pipetting
Mix
Incubate the brain section(s) Duration06:00:00 with the AcX solution at room temperature.
6h
Incubation
Pipetting
Overnight
Day 2: Sample gelation (4-fold expansion)
Day 2: Sample gelation (4-fold expansion)
3h 21m
3h 21m
Wash sample 2 x 15 mins with Amount1 mL of 1x PBS.
Pipetting
Wash
Wash sample Duration00:15:00 with Amount1 mL of 1x PBS. (1/2)

15m
Wash sample Duration00:15:00 with Amount1 mL of 1x PBS. (2/2)
15m
Thaw Stock-X solution as well as 4HT, TEMED and APS stock solutions. Vortex well and keep the solutions TemperatureOn ice .
5m
Temperature
Prepare the gelation chamber for gelation. Wipe off excess 1x PBS surrounding sample with disposable wipes (e.g., Kimwipes, Kimberly-Clark). Put the Thikness25 µm tape on both sides of the tissue section(s).


5m
Pipette Amount50 µL of the Stock-X solution on top of each tissue section. Incubate the slide at room temperature for Duration00:10:00 .

10m
Incubation
Temperature
Mix the Stock-X solution and the 4HT, TEMED, and APS stock solutions at a ratio of 94:2:2:2. Vortex to obtain the gelling solution.
Note
1. Follow the exact order when mixing the gelling solution, i.e., adding the Stock-X solution first and the APS stock solution last.
2. Move to the next step immediately.

1m
Pipetting
Mix
Critical
Remove the Stock-X solution. Add Amount50 µL of the gelling solution and gently place a coverslip over the gelation chamber. Gently press to seal the coverslip and incubate at Temperature4 °C for Duration00:30:00 .


30m
Incubation
Critical
Temperature
Polymerize the gel at Temperature37 °C for Duration01:30:00 to Duration02:00:00 .

2h
Incubation
Critical
Temperature
(Alternative) Day 2: Sample gelation (6-fold expansion)
(Alternative) Day 2: Sample gelation (6-fold expansion)
4h 41m
4h 41m
Wash sample 2 x 15 mins in Amount1 mL of 1x PBS.
Pipetting
Wash
Wash sample Duration00:15:00 in Amount1 mL of 1x PBS. (1/2)
15m
Wash sample Duration00:15:00 with Amount1 mL of 1x PBS. (2/2)
15m
Thaw Stock-S solution as well as 4HT, TEMED and APS stock solutions. Vortex well and keep the solutions TemperatureOn ice .
Temperature
Prepare the gelation chamber for gelation. Wipe off excess 1x PBS surrounding sample with Kimwipe. Put the Thikness25 µm tape on both sides of the tissue section(s).


Pipette Amount50 µL of Stock-S solution on top of each tissue section. Incubate the slide at room temperature for Duration00:10:00 .
10m
Incubation
Temperature
Mix the Stock-S solution and the 4HT, TEMED, and APS stock solutions at a ratio of 96.7 : 0.3 : 1.5 : 1.5. Vortex to obtain the gelling solution.
Note
1. Follow the exact order when mixing the gelling solution, i.e., adding the Stock-S solution first and the APS stock solution last.
2. Move to the next step immediately.

1m
Pipetting
Mix
Critical
Remove the Stock-S solution. Add Amount50 µL of the gelling solution and gently place a coverslip over the gelation chamber. Gently press to seal the coverslip and incubate at Temperature4 °C for Duration00:30:00 .


30m
Incubation
Critical
Temperature
Polymerize the gel at Temperature37 °C for Duration01:30:00 to Duration02:00:00 .
3h 30m
Incubation
Critical
Temperature
Day 2: Sample homogenization
Day 2: Sample homogenization
2d 0h 15m
2d 0h 15m
Thaw trypsin stock solution and dilute it 1:10 - 1:100 in 1x PBS to prepare trypsin digestion buffer [0.025-0.25% (w/v) in 1x PBS] at 37 °C for 2-4 days with the trypsin digestion buffer freshly prepared each day.
5m
Pipetting
Mix
Critical
Remove the chamber lid with a razor blade. Trim the gels into a right trapezoid to track the orientation of the sample.


5m
Remove the sample from the slide with a paintbrush that has been wetted with a small amount of 1x PBS and transfer it to a Amount2 mL Eppendorf containing the trypsin digestion buffer.


5m
Incubate each sample with Amount1 mL of the trypsin digestion buffer at Temperature37 °C (or TemperatureRoom temperature ) Duration48:00:00 to Duration96:00:00 .
Note
If tears or wrinkles appear after the trypsin digestion, Proteinase K (ProK) digestion (8 U/mL of ProK in 1x PBS) is an alternative for stronger homogenization.

2d
Digestion
Overnight
Temperature
Day 4: Sample expansion and immobilization
Day 4: Sample expansion and immobilization
13h 24m
13h 24m
Place a large coverslip (e.g., 260461-100, Ted Pella) on a petri dish to prepare the expansion chamber.
1m
Transfer the sample onto the coverslip in the expansion chamber.


Note
It is crucial to flip the sample (so that the tissue section side of the gel is facing up) in this step to maximize the MSI signal.

1m
Critical
Wash the sample in an excess volume of 0.5x PBS for Duration00:20:00 .

20m
Wash
Wash the sample 3 x 20 mins in an excess volume of purified water until gels are fully expanded.

Wash
Wash the sample Duration00:20:00 in an excess volume of purified water until gels are fully expanded. (1/3)
20m
Wash the sample Duration00:20:00 in an excess volume of purified water until gels are fully expanded. (2/3)
20m
Wash the sample Duration00:20:00 in an excess volume of purified water until gels are fully expanded. (3/3)
20m
Keep the sample on the large coverslip and remove excess water with a dropper or pipette.


Transfer the expanded sample onto a MALDI substrate (e.g., stainless steel MALDI plate, Applied Biosystems SCIEX or ITO-coated glass slide, PL-IF-000010-P25, Hudson Surface Technology, Inc. or MALDI IntelliSlide, 1868957, Bruker) with the help of the large coverslip.


Critical
Dry-mount the sample under vacuum in a vacuum desiccator filled with Drierite (Sigma) at TemperatureRoom temperature for Duration04:00:00 to Duration12:00:00 .

12h
Overnight
(Optional) Dry the mounted samples under high vacuum (e.g., in a CentriVap Benchtop Vacuum Concentrator, Labconco) at Temperature37 °C for Duration00:02:00 to ensure complete removal of moisture.

2m
Optional
Temperature
Day 5: Matrix application (1,5-diaminonaphthalene)
Day 5: Matrix application (1,5-diaminonaphthalene)
30m
30m
Dissolve Amount50 mg of 1,5-diaminonaphthalene (DAN) in Amount2 mL of acetone.
5m
Pipetting
Mix
Aspire the DAN acetone solution onto the bottom of the sublimation flask, and blow-dry it using nitrogen to form a thin layer of white solid.
2m
Heat up the hotplate to Temperature105 °C while placing digital thermometer in contact with the bottom of the flask to monitor the temperature.
15m
Temperature
Add ice slush to the cold finger of the apparatus, to which the MALDI plate or the glass slide with the sample is adhered on the underside with copper tape.
5m
Temperature
Place the sublimation system under a vacuum of 80 mTorr using a rough pump.

1m
Sublime DAN for Duration00:02:00 .

2m
Day 5: (Optional) Matrix recrystallization
Day 5: (Optional) Matrix recrystallization
3m
3m
Add Amount2 mL of 5% isopropyl alcohol to a glass petri dish to prepare the recrystallization chamber.

1m
Pipetting
Optional
Attach the sample coated with the matrix to the cover of the recrystallization chamber and place the chamber at Temperature55 °C for Duration00:02:00 .

2m
Optional
Day 5: Mass spectrometry imaging
Day 5: Mass spectrometry imaging
To enhance signal intensity, it is recommended to increase laser power and/or the number of shots per pixel. Transfer the MALDI plate or the glass slide with the sample to a MALDI mass spectrometer of choice and start data acquisition.
Note
1. To enhance the signal, it is recommended to increase the laser power and/or the number of shots per pixel.
2. For example, a rapifleX MALDI Tissuetyper (Bruker) can be operated in negative ion reflection mode with a mass range (m/z) of 500-2000, 10 µm or 50 µm raster distance, and 200 laser shots per pixel to to acquire image data.
3. A timsTOF fleX MALDI-2 (TTF) equipped with a Smartbeam 3D 10 kHz Nd:YAG (255 nm) laser and microGRID (Bruker) can be operated in negative ion transmission mode (MALDI-1 mode) with a mass range (m/z) of 300-2500, 5 µm, 10 µm, or 20 µm raster distances, and 45, 150, and 200 laser shots per pixel, respectively, to acquire image data.
4. In general, the total image time increases for GAMSI sample because the imaging area increases proportionally to the square of the expansion factor and more time per pixel may be needed for better signal.


Analyze
Imaging
Protocol references
Chan, Y.H., Pathmasiri, K.C., Pierre-Jacques, D. et al. Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics. Nat. Commun. 15, 5036 (2024). https://doi.org/10.1038/s41467-024-49384-w
Acknowledgements
We thank all co-authors of the original GAMSI paper, including K.C. Pathmasiri, D. Pierre-Jacques, N. Tao, J.L. Fischer, E. Yang, and S.M. Cologna, for assistance with developing this protocol. We thank F. Tobias, S. Shafaie, and the Northwestern University IMSERC facility for assistance with mass spectrometry imaging. We thank the Chicago Biomedical Consortium (CBC) for access to core mass spectrometry facilities. We thank W. Wang for assistance with hydrogel reagent preparation, C.-C. (J.) Yu for assistance with expansion isotropy analysis, and S. Kwon for assistance with scientific visualization and illustration.