Biological tissues constitute a complex array of molecular components including carbohydrates, lipids, proteins, and nucleic acids. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has proven to be a versatile technique in biomedical research to reveal distribution of broad scale of compounds ranging from metabolites to proteins within biological systems. In this work, we optimized the sample preparation protocol MALDI-IMS: cryosectioning of small fresh-frozen tissues, obtaining histology reference images, homogenous matrix deposition on tissues slices using sublimation, and data processing for the comparison of phospholipids on different tissues including mouse kidney, liver and spleen.
We demonstrate the identification and localization of different classes of phospholipids at high spatial resolution of 25 μm in positive ion mode. The lipid species were identified based on accurate mass and the structures of major phospholipids were characterized on tissue using the MALDI-IMS LIFT method. Our results showed that the kidney was enriched with the most complex and abundant phospholipids including phosphatidylcholine (PC) (32:0), PC (38:6), PC (16:0/18:1), PC (36:4), PC (34:2). On the other hand, spleen was predominantly composed of PC (32:0), while the liver with PC (34:2) and PC (16:0/18:1).
High-dimensional IMS data were visualized in lower dimension through unsupervised multivariate analysis approaches (SCiLS Lab) including image segmentation and Principal component analysis (PCA): using this approach morphological features of all tissues unambiguously distinguished. Further analysis of spleen tissues at region of interests (ROI) level using supervised approach (ClinProTools) revealed heme B is uniquely localized in red pulp, supporting the fact that splenic sinusoids are engorged with blood.
Our study outcomes emphasise that the diversity and complexity in lipid composition of each tissue type indicate structural and functional similarities / variations at cellular level. It also underscores the high potential of MALDI - IMS to unravel pathophysiological changes associated with diseases, discovery of novel biomarkers, pharmaceutical research and drug development using mouse models.