In-depth phospholipid profiling of lymphoma cells by HPLC coupled with trapped ion mobility spectrometry and high-resolution tandem mass spectrometry

• isomer-selective LC-TIMS-MS/MS workflow for phospholipid profiling • mobility-resolved fragmentation improved structural lipid annotation • co-chromatography supported the identification of ether lipid isomers • characteristic t R and CCS shifts facilitated distinction of alkyl and alkenyl lipids • lipid profiling of lymphoma cells revealed 263 species including 63 resolved isomers Lipids exhibit extensive molecular diversity and structural complexity, which poses major analytical challenges for comprehensive lipidomic profiling. Phospholipids, in particular, display extensive structural diversity and isomerism. Given the limited lipidomic data available for lymphoma cells, this work focuses on comprehensive phospholipid screening, which inherently requires the characterization of isomeric species, including plasmalogens that have been implicated in oxidative stress and ferroptosis-related cell death. Therefore, we present an efficient isomer-selective workflow based on reversed-phase liquid chromatography (RPLC) coupled to trapped ion mobility spectrometry (TIMS) and high-resolution tandem mass spectrometry (HR-MS/MS). High-confidence structural lipid annotation is achieved through the integrated evaluation of chromatographic retention time ( t R ) , exact mass-to-charge ratio ( m / z ), collision cross section (CCS) and mobility-resolved MS/MS data. Applied to human lymphoma cell lipid extracts, the workflow enabled confident identification of 263 individual lipid species spanning 10 phospholipid and 2 sphingolipid subclasses, including the resolution of 63 isomeric species at the fatty-acyl compositional level. The multidimensional approach allowed partial discrimination of fatty-acyl compositional, sn - and double bond positional isomers. Notably, characteristic deviations in both retention time and ion mobility were observed for plasmalogens relative to alkyl-ether linked phospholipids, reflecting the unique physicochemical properties of the vinyl-ether linkage. These systematic offsets enabled confident plasmalogen assignment in representative cases, supported by authentic standards, co-chromatograms and mobility-resolved fragmentation data. Collectively, this streamlined analytical platform markedly expands phospholipidome coverage and provides enhanced structural resolution of complex lipid mixtures.