Revolutions in lipid isomer resolution: application of ultra-high resolution ion mobility to reveal lipid diversity

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry ap-proaches, leading to a significant underestimation of structural diversity within the lipidome. While ion-mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require resolving power beyond the capabilities of conventional platforms. Here we present the application of high-resolution travelling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single car-bon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans) or, for glycerolipids, (iii) the rela-tive substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility sepa-ration allowed identification of carbon-carbon double bond position and sn-position, enabling confident interpreta-tion of variations in mobility-peak abundance. To demonstrate the applicability of this method, double bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of six isomers. These findings suggest that ultra-high resolution ion-mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion-activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.