Enabling spatial analysis of lipids in pbs-pfa perfused/oct-embedded mouse brain glioblastoma: a high-resolution mass spectrometry imaging approach with an ap-maldi-orbitrap

This study utilized an atmospheric-pressure matrix-assisted laser desorption/ionization (AP- MALDI) source coupled to an Orbitrap Exploris 120 mass analyzer to perform high-resolution mass spectrometry imaging (MSI) specifically targeting small molecules in glioblastoma-bearing mouse brain tissues [1]. The present approach was applied to brain tissues that were previously treated with a routinely used pharmacological preprocessing, including PBS perfusion, paraformaldehyde (PFA) fixation, and embedding in Optimal Cutting Temperature (OCT) compound. Coronal sections of 20 μm were then prepared, mounted on glass slides, and stored. Although widely employed in pharmacological workflows, the components of OCT are known to markedly suppress ionization introducing background signals, while PFA fixation and PBS rinsing can alter molecular availability and reduce MSI signal intensity. However, to enable the MSI analysis of the so-treated biological material, tissue sections were gently lifted from storage, rinsed in distilled water to remove residual embedding media, and transferred to conductive indium tin oxide (ITO)–coated slides. This repurposing approach aligns closely with the 3Rs principles (Replacement, Reduction, and Refinement), as it allows for the reuse of archived biological samples and minimizes the need for further animal sacrifices [2]. Sublimation-based matrix deposition of 2,5-dihydroxybenzoic acid (DHB) produced fine and uniform crystals, while preserving analyte localization [3]. Subsequent AP-MALDI-MS acquisition generated full-scan mass spectra across x–y coordinates, allowing the construction of detailed, color-coded 2D molecular ion maps. Despite using pre-treated samples, the protocol successfully enabled the detection and spatial mapping of tumor-associated lipids both within the tumor core and its surrounding tissue [4,5]. Lipid identification was performed by matching accurate mass data with entries from online databases such as HMDB, Metabolomics Workbench, and LipidMaps. Co-localization plots highlighted molecular distribution patterns, revealing distinct lipid enrichment within neoplastic areas. This work shows that OCTembedded brain tissues can be repurposed for MSI via a streamlined protocol, eliminating the need for extra animal experiments. The coupling of an APMALDI source with an Orbitrap analyzer enabled high- accuracy spatial mapping of tumour metabolites, regardless the use of well-known interference compounds in the tissue preparation protocols. Further studies will compare treated and untreated tumour sections to reveal metabolic shifts induced by therapy. References: [1] Bernhard Spengler et al; Brazilian Journal of Analytical Chemistry, 2023, 10, Issue 38, pp 128-13. [2] Enrico Maestri. BioTech, 2021, 10, 9. [3] Yanbo Wei et al; Analyst, 2015, 140, 1298. [4] Amanda Rae Buchberger et al; Analytical Chemistry (2018) 90 (1), 240-265. [5] Richard J.A. Goodwin. Journal of Proteomics 75 (2012) 4893 – 4911.