TITLE: NEDC matrix application for metabolite imaging using MALDI-MSI
AUTHORS: Antonia Fecke, Karl W Smith, Siva Swapna Kasarla, Philipp Bäuml, Prasad Phapale

[DESCRIPTION]
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a analytical technique more than 30 years ago. Originally developed as a method to analyze large biomolecules like proteins or peptides, MALDI-MS has over the years found a new application in the field of biological tissue imaging (MALDI-MSI). 
MALDI-MS has been utilized as an imaging tool for biological tissues, giving insight into the spatial distribution of molecules within complex tissue samples. Metabolomics research, which focuses on the study of metabolites present in a biological system, can benefit from this technology.
Small molecules such as metabolites or lipids can be analyzed under the aspect of spatial distribution across tissue sections. This enables the investigation of metabolic pathways, biomarkers or the correlation of metabolic changes with specific tissue regions, uncovering potential links between metabolite variations and physiological or pathological conditions.
Despite its potential, untargeted metabolomics using MALDI-MSI still faces several limitations. One major challenge is the diversity of matrices and protocols which are used to enhance ionization efficiency and the lack of information on metabolome coverage within different types of methods. One matrix of interest is NEDC (N-(1-napthyl)ethylendiamine dihydrochloride), which has gained recognition for its efficiency to visualize a broad range of metabolites.
This protocol provides a guide on how to prepare fresh frozen tissue samples for MALDI-MSI of metabolites using the NEDC matrix. It covers cryosectioning and preparation of tissue samples and provides a method for NEDC matrix application using the Sunchrom Sprayer.





[STEPS]
SECTION: Matrix Application
None. Matrix Application for MALDI-MSI
SECTION: Sample preparation and cryosectioning
1. Remove fresh frozen tissue sample stored at -80°C and place in cryostat at -20°C (kidney)/ -17°C (liver)/ -22°C (brain)
SECTION: Sample preparation and cryosectioning
2. Mount the sample on chuck by pipetting small droplets of water and placing the sample on top until the water is frozen
SECTION: Sample preparation and cryosectioning
3. Make sections of 12 µm thickness
SECTION: Sample preparation and cryosectioning
4. Collect the sections on an ITO slide (for MALDI-MSI) or a glass slide (for H&E staining) and thaw mount the tissue onto the slide byy warming the slide with the back of your hand
SECTION: Sample preparation and cryosectioning
5. Perform serial sectioning and xcollect the samples alternately on ITO and glass slides to allow for localzation and orientation of each ion image generated in MALDI-MSI to its histological counterpart
SECTION: Sample preparation and cryosectioning
6. Place the slides in a slide holder and store at -80°C
SECTION: Matrix application
7. Dry the sample in a vaccuum dessiccator at -0.1 MPa until dry (20-25 min)
SECTION: Matrix application
8. Prepare N-(1-naphtyl)ethylenediamine hydrochloride (NEDC) at a concentration of 7 mg/mL in MeOH:ACN:H2O (70:25:5)
SECTION: Matrix application
9. Turn on sprayer, pump (35 psi) and the connected computer
SECTION: Matrix application
10. Take 250 µL syringe, fill it with MeOH and rinse the lines of the sprayer manually
SECTION: Matrix application
11. Open the Suncollect software and enter the sprayer settings and coordinates of your tissue (based on the size of your sample)
SECTION: Matrix application
12. Fill the syringe with the prepared NEDC (solution (overfill), remove airbubbles and connect the syringe to the dispenser
SECTION: Matrix application
13. Manually press the syringe until you have removed remaining airbubbles in the lines (the spray will stop shortly if there's an airbubble)
SECTION: Matrix application
14. Place your tissue
SECTION: Matrix application
15. In the suncollect software select your syringe size and set it to filled
SECTION: Matrix application
16. Prepare your sprayer method. For this protocol you will need four separate methods (Layer 1-3, Layer 4-6, Layer 7-9 and Layer 10-21).
 
SECTION: Matrix application
17. Start the spraying method. While spraying, watch the syringe and refill inbetween the layers if empty. While refilling, also set the syringe status to filled again in the software
SECTION: Matrix application
18. After spraying, remove the tissue
SECTION: Matrix application
19. Clean you sample holder and fill the syringe again with MeOH to manually clean the lines
SECTION: Matrix application
11.4. Sprayer setting:
  

 

SECTION: Matrix application
16.1. Sprayer method
 

SECTION: Consumables
20. 
 
  
  Cryostat
  
  
  Cm 1860, Leica, Nußloch, Germany
  
  
  
  Blades
  
  
  DB80 LX Low Profile Microtome
  Blades, Leica, Nußloch, Germany
  
  
  
  ITO-slides
  
  
  Part No. CG-90IN-S115, Delta
  Technologies, Loveland, USA
  
  
  
  Glass slides
  
  
  Micro Slides 2948-75X25, Corning
  Incorporated, NY, USA
  
  
  
  N-(1-naphtyl)ethylenediamine
  hydrochloride
  
  
  CAS: 1465-25-4, PCode: 1003246811,
  Merck, St Louis, USA
  
  

 
SECTION: Matrix application
11.3.  
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11.1.  
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11.2.  
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15.1.  
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15.2.  
SECTION: Matrix application
17.1.