A Novel Lipidomics Workflow for Improved Human Plasma Identification and Quantification Using RPLC-MSn Methods and Isotope Dilution Strategies

Lipid identification and quantification are essential objectives in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity, and their dynamic range. In this work, we developed a tailored method for profiling and quantification combining (1) isotope dilution, (2) enhanced isomer separation by C30 fused-core reversed-phase material, and (3) parallel Orbitrap and ion trap detection by the Orbitrap Fusion Lumos Tribid mass spectrometer. The combination of parallelizable ion analysis without time loss together with different fragmentation techniques (HCD/CID) and an inclusion list led to higher quality in lipid identifications exemplified in human plasma and yeast samples. Moreover, we used lipidome isotope-labeling of yeast (LILY)—a fast and efficient in vivo labeling strategy in Pichia pastoris—to produce (nonradioactive) isotopically labeled eukaryotic lipid standards in yeast. We integrated the 13C lipids in the LC-MS workflow to enable relative and absolute comp...