In-Source Fragmentation Annotation in Sterol Mass Spectrometry Imaging

Spatial biology has emerged as a pivotal area in many life science fields, with mass spectrometry imaging (MSI) becoming a cornerstone for molecular imaging. Among recent advancements to increase sensitivity, MALDI-2 technology has significantly expanded the molecular space accessible to MSI, increasing the ion yields of neutral metabolites, such as sterols. Sterols have recently taken center stage in numerous (patho-) physiological processes, including neurodegenerative diseases that have attracted significant scientific interest. However, in-source fragmentation (ISF) poses a substantial challenge for accurate biological interpretation of mass spectrometric data. In this study, we observed and investigated the ISF of cholesterol during MSI under MALDI and MALDI-2 conditions. Using a murine intervention model, we demonstrate how ISF can compromise the accuracy of biological interpretations, potentially leading to significant misinterpretations. Our study underscores the critical need to address ISF to ensure accurate molecular annotation, particularly through tandem mass spectrometry of in-source fragments. This is especially important when using MALDI-2 techniques. Furthermore, we introduce a high-resolution (5 μm) MSI technique, enabling the precise spatial analysis of cholesterol distribution.