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.