May 19, 2025
Deposition of NEDC Matrix using TM Sprayer for High Resolution MALDI Analysis of Metabolites
- Ali Zahraei1,
- Martin Dufrense1,
- Jeff Spraggins1
- 1Vanderbilt University
- VU Biomolecular Multimodal Imaging Center / Spraggins Research GroupTech. support email: [email protected]
Protocol Citation: Ali Zahraei, Martin Dufrense, Jeff Spraggins 2025. Deposition of NEDC Matrix using TM Sprayer for High Resolution MALDI Analysis of Metabolites . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq65j5lk5/v1
Manuscript citation:
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 to map primary small molecules in negative mode at 10 μm spatial resolution
Created: March 28, 2025
Last Modified: May 19, 2025
Protocol Integer ID: 125690
Keywords: Metabolites, MALDI IMS, NEDC, high resolution maldi imaging mass spectrometry, imaging mass spectrometry, tm sprayer for high resolution maldi analysis, mass spectrometry, best lateral resolution of metabolite, analyte extraction, high resolution maldi analysis, analyte extraction from tissue, using tm sprayer, analyzing low molecular weight ion, htx m5 tm sprayer, automated robotic sprayer, deposition of nedc matrix, robotic sprayer, low molecular weight ions due to challenge, tissue sections for negative ion mode, metabolite
Funders Acknowledgements:
NIH
Abstract
MALDI imaging mass spectrometry (IMS) encounters difficulties in effectively analyzing low molecular weight ions due to challenges with sensitivity and spatial resolution, limiting the ability to perform a detailed characterization. This protocol describes an automated robotic sprayer (HTX M5 TM sprayer) to deposit N-(1-naphthyl) ethylenediamine hydrochloride (NEDC) onto tissue sections for negative ion mode
analyses or high resolution MALDI imaging mass spectrometry (IMS). We have optimized a method
for matrix application to maximize analyte extraction from tissue and minimize delocalization
for the best lateral resolution of metabolites.
Troubleshooting
Autofluorescence Scan
Remove slides from freezer (-80C) and place in a desiccator for 30 min.
Collect autofluorescence scan using a Zeiss AxioScan Z1: (https://www.protocols.io/edit/autofluorescence-microscopy-data-acquisition-b39kqr4w)
TM Sprayer Setup
Set nitrogen flow to 10 psi. turn on TM sprayer.
Open HTX software.
Set nozzle temperature to 55 oC, and heated stage temperature to 30 oC.
Clean sample loop with 3mL of methanol with sprayer set to "load". Switch to "spray" and allow to spray for 2 minutes.
Make the matrix solution at a concentration of 14 mg/mL in a solution of 90% methanol. Sonicate solution for 2 min.
Switch sprayer to "load" and load sample loop with 2 mL of matrix solution using a syringe.
Switch sprayer to "spray" and allow to spray for ~1 min. Check that matrix is spraying by placing a glass slide under the nozzle.
Tape slide onto heated stage, and adjust the spray area in the HTX software.
Set the method in the HTX software:
1350 mm/min nozzle velocity
2.0 mm track spacing
0.05 mL/min flow rate
CC pattern
14 passes
40 mm nozzle height
2 seconds drying time
Ensure that the method is highlighted. Press start under the Cycle tab. Click Continue. Wait for purge delay and confirm that the nozzle is moving.
When matrix deposition is finished, remove the slides and store them in a slide box. Press "valve load confirm" in the software.
Celanup
Set both TM sprayer nozzle and stage temperatures to 30 oC.
With sprayer set to "load", load loop with 3 mL of methanol methanol. Set sprayer to "spray" and allow to flow for ~5 min.
Spray the nozzle tip with methanol and acetonitrile, not touching the tip.
Clean the stage with Kimwipes tissue that is sprayed with methanol.
Shut down HTX software and close the computer. Turn off the TM sprayer.
Shut off the nitrogen flow.
Dispose of syringes in biohazard container.