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.
Highlights
The range of applications of mass spectrometry imaging (MSI) has grown exponentially over the last decades [1, 2]
This study consists of three parts: (1) the optimization of a sample preparation method of undecalcified bone tissue for MSI, focused on the embedding material and sectioning method; (2) a targeted approach measuring drug concentrations in rat and human bone tissue, and on the detection of methadone and its metabolites to determine the limit of detection (LOD) and detection of these molecules in dosed bones; and (3) an untargeted approach to select the most suitable matrix for measuring lipids in mouse bone tissue
This study describes the optimization of the sample preparation protocol for matrix-assisted laser desorption/ ionization (MALDI)-MSI of undecalcified bone tissue in close contact with soft tissue like bone marrow or muscle tissue
Summary
The range of applications of mass spectrometry imaging (MSI) has grown exponentially over the last decades [1, 2]. A main advantage of MSI is the possibility to detect a wide range of molecules and visualize their distributions without the need for targeted labels while maintaining sample integrity [1]. The development of matrix-assisted laser desorption/ ionization (MALDI)-MSI has contributed to a broader application field for MSI, due to the soft ionization of molecules and the broader molecular weight range than previous ionization techniques, for example, secondary ionization mass spectrometry (SIMS) [1]. The sample preparation workflow for MALDI-MSI is highly critical for the quality and reliability of the MSI measurements. The quality and reliability depend on the complexity of the tissue of interest. The sample preparation will affect, among others, sample integrity, the ionization efficiency of molecules, and local ion suppression due to salts and endogenous compounds in the tissue
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