Mobility‐Modulated Sequential Dissociation Analysis Enables Structural Lipidomics in Mass Spectrometry Imaging

Abstract Spatial lipidomics based on mass spectrometry imaging (MSI) is a powerful tool for fundamental biology studies and biomarker discovery. But the structure‐resolving capability of MSI is limited because of the lack of multiplexed tandem mass spectrometry (MS/MS) method, primarily due to the small sample amount available from each pixel and the poor ion usage in MS/MS analysis. Here, we report a mobility‐modulated sequential dissociation (MMSD) strategy for multiplex MS/MS imaging of distinct lipids from biological tissues. With ion mobility‐enabled data‐independent acquisition and automated spectrum deconvolution, MS/MS spectra of a large number of lipid species from each tissue pixel are acquired, at no expense of imaging speed. MMSD imaging is highlighted by MS/MS imaging of 24 structurally distinct lipids in the mouse brain and the revealing of the correlation of a structurally distinct phosphatidylethanolamine isomer (PE 18 : 1_18 : 1) from a human hepatocellular carcinoma (HCC) tissue. Mapping of structurally distinct lipid isomers is now enabled and spatial lipidomics becomes feasible for MSI.