Quantitative MALDI-MSI Workflow for Lipids with a LC-MS-Based Quality Control Pipeline

Mass spectrometry imaging (MSI) has established itself as a major analytical method in the spatial analysis of biological tissue. Besides qualitative mapping of analyte distributions, recent developments aim to achieve quantification of target analytes. After successful spatial quantification of pharmaceuticals, the field is now shifting toward endogenous biomolecules such as lipids and metabolites. Here, we present a quantitative MSI (qMSI) workflow for endogenous lipids employing matrix-assisted laser desorption/ionization (MALDI) and stable isotope labeled standards (SILS) integrated into a quality control pipeline utilizing quantitative reversed-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS). We thoroughly characterized the standard deposition of 13 lipid SILS with a focus on homogeneity and detection errors on mouse heart septa with intraday coefficient of variations (CVs) of ≤15%. Unlike RP-LC-MS/MS for which CV values increase mainly for lowly abundant lipids, variations of lipids in MALDI-MSI are affected by concentration as well as lipid class. Additionally, we compared lipid annotations based on MALDI-MSI and RP-LC-MS/MS and correlated these annotations between the methods based on quantification results. This method comparison revealed that most polar lipids such as PCs or SMs are captured in both MALDI-MSI and RP-LC-MS/MS, whereas less polar compounds such as TGs exhibit limited overlap. These results highlight that a determination of the SILS amounts and lipid annotations via RP-LC-MS/MS is necessary to counter the increase variability during quantitative MALDI-MSI. To demonstrate the analytical capabilities of the quantitative MALDI-MSI pipeline combined with RP-LC-MS/MS quality control steps, we analyzed mouse kidney from a two-hit heart failure with preserved ejection fraction model and achieved good comparability between RP-LC-MS/MS and MALDI-MSI quantitation results. Our data suggest increased ether lipid concentrations in obese mice kidney, likely to cope with increased oxidative stress induced by the progressing disease state of the mice.