Tandem Mass Spectrometry Imaging of Low-Abundance Ether Phospholipids Guided by Bulk Lipidomics Analysis

A central challenge in mass spectrometry imaging (MSI) of lipids is detecting and identifying species overshadowed by higher abundance isobaric lipids. Although tandem mass spectrometry can differentiate many lipid isobars and some isomers, the diversity and heterogeneity of lipids observed in MSI experiments necessitate the development of targeted approaches to differentiate and localize species with an overlapping m/z. To address this challenge, we leverage the fast acquisition speed of multiple reaction monitoring (MRM) to identify and localize hundreds of lipids in a single MSI experiment. We present a workflow to generate comprehensive, system-specific, and information-rich MRM transition lists for MSI and use them to examine the spatial localization of specific lipid classes with nanospray desorption electrospray ionization (nano-DESI) MSI in MRM mode. The lists are generated by integrating information from the LIPID MAPS database with data-dependent acquisition (DDA) and filtering by examining MRM signals of the corresponding tissue lipid extract. Using 165 MRM transitions in a single nano-DESI MSI experiment, we examined the localization of plasmalogen species, their isomers, and corresponding isobars in mouse brain tissue with sn-chain-level annotation. This workflow, which can be readily adapted to other lipid classes and tissue types, establishes MRM-MSI as a powerful strategy for mapping lipid targets in complex tissues.