Materials and Methods
Tissue preparation
All experiments on animals were performed according to the respective
Polish and European Communities Council Directives (86/609/EEC) and were
approved by the local Ethics Committee (approval number 137/2018). The
tissues were obtained as spared from control animals used during other
experiments, according to the so‐called 3R (replacement/reduction/
refinement) rule. Immediately after isolation, the rat spinal cords were
flash frozen and kept in a liquid nitrogen storage dewar (LS750,
Worthington Industries, Columbus, OH, USA) until assayed. Approximately
1 h before cutting, the tissue was transferred to the cryomicrotome
chamber (Cryotome FSE, Thermo Fisher Scientific, Chesire, UK). Both, the
chamber, and the specimen temperatures were set to −15°C. Slices were
cut at 12 µm slices, and immediately thaw‐mounted on the indium-tin
oxide (ITO) glasses (Bruker‐Daltonics, Bremen, Germany). The tissue
slices were then vaccum-dried for about 45 min. Prepared samples were
stored in a laboratory freezer at −80°C (NU9483, NuAire Inc., Plymouth,
MN, USA) in the hermetically sealed boxes. Just before analysis, the
samples were warmed up to an ambient temperature in a vacuum desiccator
to prevent moisture condensation on the sample’s surface and dried again
for about 45 min. Before each matrix deposition, an optical image (600
dpi) of the tissue cross‐sections with marked fiducials was recorded.
Additionally, as suggested by Angel et al.19, before
analysis in both modes (pos./neg.), tissue washing by 50 mM ammonium
acetate pH = 6.5 by 10 s has been tested. This procedure is suggested,
especially in the negative ionization mode, as a way to improve the
sensitivity of the measurements.
MALDI matrices:
During the experiments, the following chemicals were used: solvents: ACN
and MeOH (J.T. Baker, Amsterdam, Netherlands), both at the HPLC gradient
grade, ethanol (Avantor, Poland) chloroform, ultrapure water
(Sigma-Aldrich/Merck, Germany). Matrices: 2,5-dihydroxybenzoic acid
(DHB), trifluoroacetic acid (TFA), norharmane, N-(1-naphthyl)
ethylenediamine dihydrochloride (NEDC) all from Sigma-Aldrich/Merck
(Darmstadt, Germany).
Matrix application:
The matices were applied by a SunCollect® device (SunChrom GmbH,
Friedrichsdorf, Germany). The flow rate of the matrix solution changed
among layers, according to the manufacturer recommendations: 10 µL/min
for the first layer, 20 µL/min for the second layer, 30 µL/min for the
third layer, 40 µL/min for the fourth layer, and 60 µL/min for all
following layers. The nozzle applied the matrix solution with a line
distance of 2 mm, and the speed 600 mm/min. For each matrix, a different
number of layers was applied. The Z value – the position of the
spraying nozzle above the tissue surface was also tested. Z=1 means the
highest possible (about 50 mm) position above the tissue, whereas Z=25
means about 25mm above the tissue and is determined as the “extraction
mode”. Four different numbers of layers and four different nozzle
heights were tested for each matrix.
MALDI measurements
Matrix‐coated sections were subjected to imaging experiments using the
MALDI–TOF/TOF UltrafleXtreme MS (Bruker-Daltonics, Bremen, Germany)
with a Smartbeam II™ laser operating at 2 kHz. All following MS
parameters underwent initial, multistep optimization. Ions were
accelerated at 25 kV with a pulsed ion extraction of 120 ns and ion
suppression up to 100 Da. Spectra were recorded in positive and negative
ion modes with reflectron, within an 200–3000 m/z range. They were
externally calibrated with Peptide Calibration Standard II
(Bruker-Daltonics, Bremen, Germany) and known matrix ions, e.g. 273.0399
m/z for DHB matrix ([2DHB-2H2O+H]+ion) and 229.0533 m/z for 9AA matrix ([9AA+Cl]-ion). A raster width of 200 μm was applied to all samples. In total, 400
shots were collected from each ablation point with 20 shots at the
raster spot, and the laser focus diameter was set to “3_medium”.
FlexControl version 3.4 (Bruker-Daltonics, Bremen, Germany) was employed
for spectra acquisition, and FlexImaging, version 4.0 was used for data
processing and the creation of molecular images. Mmass software (version
5.5.0, Open-Source software developed by Martin Strohalm, Academy of
Sciences, Prague, Czech Republic) was used for the spectra
analysis20.