Tracing <em>de novo</em> Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (<1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.