Introduction
Sample preparation is one of the most crucial steps in every analytical
procedure. Optimization of this part of the experiment is usually the
most time-consuming task. Nevertheless, once successfully optimized, the
procedure allows for certain and repetitive analyses. Sample preparation
for MALDI-MSI is connected to the application of a special matrix before
the analysis. The scientists beginning their adventure with this complex
and powerful technique may be overwhelmed by the variety of matrices,
solvents, concentrations, the ways of their applications, and the lack
of widely available knowledge about the influence of these parameters on
the final results. Some data can be found in the research articles,
technical reports, or protocols provided by the equipment manufacturers,
but usually, it is not easy to find a comprehensive recommendation on
which matrix and sample preparation strategy to select.
In our study, we decided to use common matrices like
2,5-dihydroxybenzoic acid (DHB), 9-aminoacridine (9AA),
1.5-diaminonaphtalene (DAN), and not as well-known matrices as
norharmane and N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) to
compare their ability to visualize lipids and in some cases (NEDC
matrix) small molecules.
To apply matrices, we used SunCollect® system, which utilizes a
high-performance pneumatic sprayer. It is recognized as a wet-interface
system, and it is worth to mention that its performance may be different
form the systems based for example on sublimation process1 (see: supplementary materials from mentioned
article). In this device, the matrix solution could be sprayed in
several layers over the tissue sample and from different heights over
the sample. The higher position (e.g., topmost position Z=1) produces
the driest vapor resulting in the finest matrix crystals size. At the
lowest position (Z = 25 mm), the wetter, atomized spray will attain the
target. It means that the matrix solution could penetrate the tissue
sample and allows to observe the substances that must be extracted from
the tissue. In our study, four different positions of the spraying
nozzle above the sample surface and the influence of the multiple matrix
layers on the quality of obtained MS spectra were examined for each
matrix.
DHB matrix is the most common one for the analysis in the positive
ionization mode. In the literature different matrix concentration (from
7 mg/ml2, to even 50 mg/ml3 could be
applied. Different solvents are used as well. Mainly methanol
(MeOH)4 and acetonitrile (ACN) 5,6but also ethanol7 and mixtures of chloroform and
methanol8 are used. 9AA is a popular matrix for the
negative ionization mode. In this case, concentration between 2 mg/ml9 and 10 mg/ml10 are used, but the
most commonly selected is 7 mg/ml (like in11–13).
Regarding the solvents, mainly aqueous solutions of methanol and ethanol
are used in different concentrations. Norharmane and NEDC are still
gaining their popularity so, there is no such diversity in their use. In
both cases, 7mg/ml is the most popular
concentration13. Regarding norharmane for SunCollect®
application, the concentration of 6 mg/ml and the solution of
chloroform: MeOH:H2O, 1:2:0.8 (v/v/v) is used since in
given volumes ratios, the solution creates a homogenous mixture, and
separation of water and chloroform is not observed. Additionally,
1.5-DAN (25 mg/ml, in ACN:H2O, 1:1,
v/v)14 and 1.5 DAN hydrochloride proposed by Liu et
al.15 were tested.
The cross-section of a rat spinal cord was chosen as a tissue model,
representing the features of the central nervous system. Particularly,
its simple structural division into a gray and white matter should be
easily recognizable on MSI tissue scans. Additionally, the slices
obtained from this tissue are small and thus convenient for the fast MSI
measurements. Thickness of the slides was typical for most MALDI-MSI
experiments and was set to 12 µm. To compare different matrices, we
decided to choose several different peaks representing lipids from the
spinal cord’s gray and white matter, well known in the
literature16–18. Such choice allows for visualization
of the structures and comparing the quality of obtained results between
different matrices. Additionally, the care was taken to choose
intensive, as well as relatively small, peaks to show their behavior in
the broad intensity range (see Fig.1). Finally, the parameters of chosen
peaks, especially their intensities, were used for the comparison.