Sample preparation of bone tissue for MALDI-MSI for forensic and (pre)clinical applications

Abstract

In the past decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been applied to a broad range of biological samples, e.g., forensics and preclinical samples. The use of MALDI-MSI for the analysis of bone tissue has been limited due to the insulating properties of the material but more importantly the absence of a proper sample preparation protocol for undecalcified bone tissue. Undecalcified sections are preferred to retain sample integrity as much as possible or to study the tissue-bone bio interface in particular. Here, we optimized the sample preparation protocol of undecalcified bone samples, aimed at both targeted and untargeted applications for forensic and preclinical applications, respectively. Different concentrations of gelatin and carboxymethyl cellulose (CMC) were tested as embedding materials. The composition of 20% gelatin and 7.5% CMC showed to support the tissue best while sectioning. Bone tissue has to be sectioned with a tungsten carbide knife in a longitudinal fashion, while the sections need to be supported with double-sided tapes to maintain the morphology of the tissue. The developed sectioning method was shown to be applicable on rat and mouse as well as human bone samples. Targeted (methadone and EDDP) as well as untargeted (unknown lipids) detection was demonstrated. DHB proved to be the most suitable matrix for the detection of methadone and EDDP in positive ion mode. The limit of detection (LOD) is estimated to approximately 50 pg/spot on bone tissue. The protocol was successfully applied to detect the presence of methadone and EDDP in a dosed rat femur and a dosed human clavicle. The best matrices for the untargeted detection of unknown lipids in mouse hind legs in positive ion mode were CHCA and DHB based on the number of tissue-specific peaks and signal-to-noise ratios. The developed and optimized sample preparation method, applicable on animal and human bones, opens the door for future forensic and (pre)clinical investigations.

Keywords: Bone tissue; MALDI; Mass spectrometry imaging; Sample preparation.

Fig. 1

Analytical workflow for the analysis of bone tissue with MALDI-MSI. Samples were collected and stored in the freezer until further use. Samples were embedded in 20% gelatin with 7.5% CMC (w/v), sectioned at 12 μm with the support of double-sided tapes, and mounted with the other side of the tape on a glass slide such that the tissue was available for analysis. The matrix was applied on the sample via sublimation. After matrix sublimation, the MALDI-MSI measurement was performed, during which a mass spectrum per pixel is obtained for the region of interest. After data processing and analysis of these spectra, for example, intensity and signal-over-noise (S/N) values could be obtained and distribution images of selected m/z values could be created that can provide information about the location of these molecules

Fig. 2

Calibration series for methadone and EDDP. a Longitudinal bone section spiked with methadone dilution series region of interest (ROI) in blue. (b) MALDI-MSI image of methadone (m/z = 310.21) and (c) EDDP (m/z = 278.18). The distribution images are total ion current (TIC)-normalized. Scale bar shows relative intensities

Fig. 3

Distribution images of methadone and EDDP in a rat femur dosed with methadone. a Longitudinal section of rat femur with region of interest (ROI) in blue. b MALDI-MSI distribution image of methadone (m/z 310.21). c MALDI-MSI distribution image of EDDP (m/z 278.18). The distribution images are total ion current (TIC)-normalized. Scale bar shows relative intensities

Fig. 4

Distribution images of methadone and EDDP in a human clavicle from someone who overdosed on methadone. a Cross section of the clavicle with the region of interest (ROI) in blue. b MALDI-MSI distribution image of methadone (m/z 310.21). c MALDI-MSI distribution image of EDDP (m/z 278.18). The distribution images are total ion current (TIC)-normalized. Scale bar shows relative intensities

Fig. 5

Overlay images of selected m/z values for CHCA, DHA, DAN, DHB, and norharmane in positive ion mode measured with MALDI-MSI of mouse hind legs for the untargeted detection of lipids. The m/z values were selected to show the similarities and differences between the matrices in terms of desorption and ionization efficiency. a Optical scan of the section with the measured area indicated with a black line. Overlay images of the distributions of selected m/z values in the range 200–500 (b), 500–600 (c), and 700–800 (d). The distribution images are total ion current (TIC)-normalized and the intensity scale shows the maximum relative intensity of the specific m/z value (for tentative identifications of the selected m/z values, see ESM Table S3)