Revolutions in Lipid Isomer Resolution: Application of Ultrahigh-Resolution Ion Mobility to Reveal Lipid Diversity

Abstract

Many families of lipid isomers remain unresolved by contemporary liquid chromatography−mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon−carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon−carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and snposition 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 up to six isomers. These findings suggest that ultrahigh-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.

MobiLipid: A Tool for Enhancing CCS Quality Control of Ion Mobility–Mass Spectrometry Lipidomics by Internal Standardization

“…Especially in the case of TWIM-MS lipidomics applications, it has been shown that either a calibration , or an external postcalibration correction using (class-specific) lipids leads to an improved trueness of CCS values compared to DT CCS and/or published TW CCS values. Moreover, high-resolution IM analyzers, including structures for lossless ion manipulation (SLIM) and cyclic IMS offer new possibilities for challenging lipid isomer separations for which a CCS calibration provides new challenges. Recent studies revealed that TIM CCS values of lipids generally show good agreement with DT CCS when relying on external calibration using the same calibrant ions (ESI Tune Mix calibration standard). , However, a CCS recalibration of each analytical run is recommended in this type of workflow.…”

“…■ EXPERIMENTAL SECTION Sample Preparation. As samples, an unlabeled and a uniformly 13 C-labeled ethanolic yeast (Komagataella phaff ii) extract pellet 16 were provided by Isotopic Solutions (Vienna, Austria). The yeast extract pellets were re-extracted using a two-phase lipidomics extraction based on methyl tert-butyl ether (MTBE) modified from Matyash et al 26 More details on the extraction procedure can be found in the Supporting Information.…”