High-resolution AP-MALDI-orbitrap mass spectrometry imaging: spatial metabolite profiling in murine glioblastoma brain tissue

Atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) coupled with an Orbitrap Exploris 120 mass analyzer was optimized for high-resolution mass spectrometry imaging (MSI) of small molecules in glioblastoma-bearing mouse brain tissues [1]. Brain samples had previously undergone standard pharmacological procedures, including PBS perfusion, paraformaldehyde (PFA) fixation, and embedding in Optimal Cutting Temperature (OCT) compound. From these, 20 μm-thick coronal sections were cut, mounted on standard glass slides, and stored. While common in pharmacological studies, these treatments are not optimal for imaging applications due to their well-known ion suppression effects. For this study, the stored sections were carefully detached, gently washed in distilled water and remounted onto indium tin oxide (ITO)- coated conductive slides. This repurposing strategy is fully in line with the 3Rs principles (Replacement, Reduction, and Refinement), enabling the reuse of archival biological material and reducing the need for additional animal sacrifice [2]. Matrix deposition via sublimation with 2,5- dihydroxybenzoic acid (DHB), yielded fine, homogeneous crystals without analyte delocalization [3]. Subsequent APMALDI acquisition produced full-scan mass spectra across x–y coordinates, enabling generation of detailed molecular ion maps in a color-coded 2D format [4]. Despite the use of pre- treated samples [5], the protocol enabled detection and spatial localization of tumour-associated lipids, both within and around the tumour mass. Lipid identities were assigned using accurate mass data matched against online databases (HMDB, Metabolomics Workbench, LipidMaps); co- localization plots illustrated molecular distribution patterns, revealing precise lipid enrichment in neoplastic regions. This study demonstrates that pre-existing OCT-embedded brain samples can be successfully repurposed for mass spectrometry imaging analyses, through a carefully optimized protocol, thus avoiding additional animal sacrifices. Furthermore, AP-MALDI coupled with an Orbitrap analyzer proves to be a robust analytical tool for high accuracy molecular mapping of tumour microenvironments. Future efforts will focus on comparative imaging of treated versus untreated tumour samples to elucidate metabolic alterations due to therapeutic treatments. 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.