biostudies-literatureUnknown14(1)Xia TA lipidome comprises thousands of lipid species, many of which are isomers and isobars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), although widely used for lipidomic profiling, faces challenges in differentiating lipid isomers. Herein, we address this issue by leveraging the orthogonal separation capabilities of hydrophilic interaction liquid chromatography (HILIC) and trapped ion mobility spectrometry (TIMS). We further integrate isomer-resolved MS/MS methods onto HILIC-TIMS, which enable pinpointing double bond locations in phospholipids and sn-positions in phosphatidylcholine. This system profiles phospholipids at multiple structural levels with short analysis time (<10 min per LC run), high sensitivity (nM detection limit), and wide coverage, while data analysis is streNature communications4263https://www.ebi.ac.uk/biostudies/studies/S-EPMC10352238biostudies-literatureDeep-profiling of phospholipidome via rapid orthogonal separations and isomer-resolved mass spectrometry.PMC10352238Xia YYin HZhang DJin XZhou FXia TGong YShi HfalseDeep-profiling of phospholipidome via rapid orthogonal separations and isomer-resolved mass spectrometry.A lipidome comprises thousands of lipid species, many of which are isomers and isobars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), although widely used for lipidomic profiling, faces challenges in differentiating lipid isomers. Herein, we address this issue by leveraging the orthogonal separation capabilities of hydrophilic interaction liquid chromatography (HILIC) and trapped ion mobility spectrometry (TIMS). We further integrate isomer-resolved MS/MS methods onto HILIC-TIMS, which enable pinpointing double bond locations in phospholipids and sn-positions in phosphatidylcholine. This system profiles phospholipids at multiple structural levels with short analysis time (<10 min per LC run), high sensitivity (nM detection limit), and wide coverage, while data analysis is stre2023-01-01T00:00:00Z2023 Jul2025-04-21T16:14:49.14Z2025-04-21T16:14:49.14ZS-EPMC103522383746055810.1038/s41467-023-40046-x